Oxidized cellulose microspheres

ABSTRACT

A process for forming microspheres is disclosed. The process includes contacting a solvent with a modified cellulose to form a solution; contacting the modified cellulose solution with at least one bioactive agent to form a discontinuous phase liquid; contacting the discontinuous phase liquid with a continuous phase liquid to form an emulsion; and contacting the emulsion with a third phase liquid to extract the solvent from the emulsion, thereby forming a plurality of modified cellulose microspheres.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional application U.S. patentapplication Ser. No. 13/903,297, filed May 28, 2013, which claims thebenefit of and priority to U.S. Provisional Application Ser. No.61/653,620, filed May 31, 2012, the entire contents of all of which areincorporated by reference herein.

BACKGROUND

Technical Field

The present disclosure relates to systems and methods for dissolvingcellulose. In particular, the present disclosure provides processes fordissolving modified cellulose.

Background of Related Art

Cellulose is the most abundant biorenewable material, andcellulose-derived products have been used in multiple industries,including manufacturing of textiles and medical devices. Apart from theuse of unmodified cellulose-containing materials (for example wood,cotton), modern cellulose technology requires extraction and processingof cellulose from primary sources using techniques that have changedvery little since the inception of the modern chemical industry.

The full potential of cellulose and cellulose products has not beenfully exploited, partially due to the historical shift towardspetroleum-based polymers, and also by the limited number of commonsolvents in which cellulose is readily soluble. Traditional cellulosedissolution processes, including the cuprammonium and xanthateprocesses, are often cumbersome or expensive and require the use ofunusual solvents, typically with a high ionic strength, under relativelyharsh conditions.

Various processes for dissolving cellulose have been previouslydisclosed. See, for example, McCormick, et al. “Solution Studies ofCellulose in Lithium Chloride and N,N-Dimethylacetamide,”Macromolecules, 1985, Vol. 18, No. 12, 1985, pp. 2394-2401; Timpa,“Application of Universal Calibration in Gel Permeation Chromatographyfor Molecular Weight Determination of Plant Cell Wall Polymers: CottonFiber,” J. Agric. Food Chem., 1991, 39, 270-275; and Strli{hacek over(c)} et al., “Size Exclusion Chromatograhy of Cellulose inLiCl/N,N-Dimethylacetamide,” J. Biochem. Biophys. Methods, 2003, 56, pp.265-279.

Improved processes for dissolving cellulose, that overcome the need forhigh thermal treatment, excessive physical manipulation (e.g.,stirring), and/or lengthy treatment periods, all of which contribute tothe degradation of the cellulose and removal of oxidized groups fromoxidized cellulose, remain desirable.

SUMMARY

In one embodiment, the present disclosure provides a process including:forming a mixture by contacting a modified cellulose with a solventunder an inert atmosphere to form a swelled modified cellulose;adjusting the mixture to a first temperature from about 115° C. to about145° C.; contacting the swelled modified cellulose with a salt under theinert atmosphere to form a modified cellulose solution; and adjustingthe modified cellulose solution to a second temperature from about 90°C. to about 120° C.

According to an aspect of the above embodiment, the first temperature isfrom about 120° C. to about 140° C.

According to an aspect of the above embodiment, the first temperature isfrom about 130° C. to about 135° C.

According to an aspect of the above embodiment, the second temperatureis from about 100° C. to about 110° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the modified celluloseis an oxidized cellulose.

According to an aspect of the above embodiment, the modified cellulosesolution includes dissolved oxidized cellulose having a degree ofoxidation from about 80% to about 120% of a degree of oxidation ofpredissolved oxidized cellulose.

According to an aspect of the above embodiment, the modified cellulosesolution includes dissolved oxidized cellulose having a molecular weightfrom about 80% to about 120% of the molecular weight of predissolvedoxidized cellulose.

According to another embodiment, the present disclosure provides aprocess including: forming a mixture by contacting an oxidized cellulosewith a solvent under an inert atmosphere to form a swelled oxidizedcellulose, the oxidized cellulose having a degree of oxidation of fromabout 0.2 to about 1.0; adjusting the mixture to a first temperaturefrom about 115° C. to about 145° C.; contacting the swelled oxidizedcellulose with a salt under the inert atmosphere to form an oxidizedcellulose solution; and adjusting the oxidized cellulose solution to asecond temperature from about 90° C. to about 120° C., wherein thedissolved oxidized cellulose has a degree of oxidation from about 80% toabout 120% of the degree of oxidation of predissolved oxidizedcellulose.

According to an aspect of the above embodiment, the first temperature isfrom about 120° C. to about 140° C.

According to an aspect of the above embodiment, the second temperaturefrom about 100° C. to about 110° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the salt is present inan amount of from about 0.1% by weight to 3% by weight of the oxidizedcellulose.

In a further embodiment, the present disclosure provides for a processincluding: forming a mixture by contacting a modified cellulose with asolvent under an inert atmosphere to form a swelled modified cellulose;adjusting the mixture to a first temperature from about 115° C. to about145° C.; contacting the swelled modified cellulose with a salt under theinert atmosphere to form a modified cellulose solution under the inertatmosphere; and adjusting the modified cellulose solution to a secondtemperature from about 90° C. to about 120° C., wherein the dissolvedmodified cellulose has a molecular weight from about 80% to about 100%of the molecular weight of predissolved modified cellulose.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the first temperature isfrom about 120° C. to about 140° C.

According to an aspect of the above embodiment, the second temperaturefrom about 100° C. to about 110° C.

According to an aspect of the above embodiment, the salt is present inan amount of from about 0.1% by weight to 3% by weight of the modifiedcellulose.

According to an aspect of the above embodiment, the modified celluloseis an oxidized cellulose.

In one embodiment, the present disclosure provides for a solution ofmodified cellulose that is formed by contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the mixture to a first temperature; contacting theswelled modified cellulose with a salt under the inert atmosphere toform a modified cellulose solution; adjusting the modified cellulosesolution to a second temperature that is lower than the firsttemperature; and contacting the modified cellulose solution with atleast one multivalent cation to form a plurality of modified celluloseparticles.

According to an aspect of the above embodiment, the contacting of theswelled modified cellulose is performed after adjusting the firsttemperature.

The present disclosure also provides a solution of modified celluloseincluding dissolved modified cellulose having a molecular weight fromabout 80% to about 100% of the molecular weight of predissolved modifiedcellulose.

In one embodiment, the present disclosure provides a process including:forming a modified cellulose solution; and contacting the modifiedcellulose solution with at least one multivalent cation to form aplurality of modified cellulose particles.

According to an aspect of the above embodiment, the forming of themodified cellulose solution includes: contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose with a salt underthe inert atmosphere to form a modified cellulose solution; andadjusting the modified cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the at least onemultivalent cation is selected from the group consisting of cations ofcalcium, barium, zinc, magnesium, chromium, platinum, and iron.

According to an aspect of the above embodiment, the process furtherincludes shearing the modified solution to form the plurality ofmodified cellulose particles.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the modified celluloseis an oxidized cellulose.

According to an aspect of the above embodiment, the plurality ofmodified cellulose particles include oxidized cellulose having a degreeof oxidation from about 80% to about 120% of a degree of oxidation ofpredissolved oxidized cellulose.

According to an aspect of the above embodiment, the plurality ofmodified cellulose particles include oxidized cellulose having amolecular weight from about 80% to about 120% of the molecular weight ofpredissolved oxidized cellulose.

In one embodiment, the present disclosure provides a process including:forming an oxidized cellulose solution; and contacting the oxidizedcellulose solution with at least one multivalent cation to form aplurality of oxidized cellulose particles having a degree of oxidationfrom about 80% to about 120% of the degree of oxidation of predissolvedoxidized cellulose.

According to an aspect of the above embodiment, the forming of theoxidized cellulose solution includes: contacting an oxidized cellulosewith a solvent under an inert atmosphere to form a swelled oxidizedcellulose having a degree of oxidation of from about 0.2 to about 1.0;adjusting the swelled oxidized cellulose to a first temperature;contacting the swelled oxidized cellulose with a salt under the inertatmosphere to form an oxidized cellulose solution; and adjusting theoxidized cellulose solution to a second temperature that is lower thanthe first temperature.

According to an aspect of the above embodiment, the first temperature isfrom about 120° C. to about 140° C. and the second temperature fromabout 100° C. to about 110° C.

According to an aspect of the above embodiment, including shearing theoxidized solution to form the plurality of oxidized cellulose particles.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the at least onemultivalent cation is selected from the group consisting of cations ofcalcium, barium, zinc, magnesium, chromium, platinum, and iron.

In one embodiment, the present disclosure provides a process including:forming a modified cellulose solution; and contacting the modifiedcellulose solution with at least one multivalent cation to form aplurality of modified cellulose particles having a molecular weight fromabout 80% to about 100% of the molecular weight of predissolved modifiedcellulose.

According to an aspect of the above embodiment, the forming of themodified cellulose solution includes: contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose with a salt underthe inert atmosphere to form an modified cellulose solution; andadjusting the modified cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the first temperature isfrom about 120° C. to about 140° C. and the second temperature fromabout 100° C. to about 110° C.

According to an aspect of the above embodiment, the process furtherincludes shearing the modified solution to form the plurality ofmodified cellulose particles.

According to an aspect of the above embodiment, the at least onemultivalent cation is selected from the group consisting of cations ofcalcium, barium, zinc, magnesium, chromium, platinum, and iron.

According to an aspect of the above embodiment, wherein the modifiedcellulose is an oxidized cellulose.

In one embodiment, the present disclosure provides a process for forminga composition including: forming a modified cellulose solution; forminga cationic composition cross-linkable with the modified cellulosesolution; and contacting the modified cellulose solution and thecationic composition at a treatment site thereby forming an ionicallycross-linked gel.

According to an aspect of the above embodiment, the formation of themodified cellulose solution includes: contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose with a salt underthe inert atmosphere to form a modified cellulose solution; andadjusting the modified cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the modified cellulosesolution has a pH from about 8.0 to about 9.5.

According to an aspect of the above embodiment, the cationic compositionis an aqueous solution of chitosan having a pH from about 2.0 to about6.0.

According to an aspect of the above embodiment, the cationic compositionis an aqueous solution of at least one multivalent cation.

According to an aspect of the above embodiment, the at least onemultivalent cation is selected from the group consisting of cations ofcalcium, barium, zinc, magnesium, chromium, platinum, and iron.

According to an aspect of the above embodiment, the process furtherincludes convergently applying the modified cellulose solution and thecationic composition onto a treatment site.

According to an aspect of the above embodiment, the modified celluloseis an oxidized cellulose.

In one embodiment, the present disclosure provides a process for forminga composition including: forming a modified cellulose solution; forminga gelation composition; and contacting the modified cellulose solutionand the composition at a treatment site thereby forming a gel.

According to an aspect of the above embodiment, the formation of themodified cellulose solution includes: contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose with a salt underthe inert atmosphere to form a modified cellulose solution; andadjusting the modified cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the gelation compositionis an aqueous solution of chitosan having a pH from about 2.0 to about6.0.

According to an aspect of the above embodiment, the modified cellulosesolution has a pH from about 8.0 to about 9.5.

According to an aspect of the above embodiment, the gelation compositionis an aqueous solution of at least one multivalent cation.

According to an aspect of the above embodiment, the at least onemultivalent cation is selected from the group consisting of cations ofcalcium, barium, zinc, magnesium, chromium, platinum, and iron.

According to an aspect of the above embodiment, gelation composition isselected from the group consisting of water, saline, phosphate bufferedsaline, and combinations thereof.

According to an aspect of the above embodiment, the gelation compositionis an aqueous solution of carboxymethylcellulose, wherein thecarboxymethylcellulose is present from about 0.5% by weight of thesolution to about 5% by weight of the solution.

According to an aspect of the above embodiment, the gelation compositionis a solution of an acrylic polymer based on at least one of methylmethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, glycerylacrylate, glyceryl methacrylate, acrylic acid, methacrylic acid,acrylamide, or methacrylamide, and combinations thereof.

According to an aspect of the above embodiment, the solution includes asolvent selected from the group consisting of acetone, ethyl acetate,dimethyl ether, and combinations thereof.

According to an aspect of the above embodiment, the gelation compositionincludes a Schiff-base compound selected from the group consisting ofamoxicillin, cephalexin, and combinations thereof.

According to an aspect of the above embodiment, the gelation compositionincludes trilysine, albumin, polyethylene glycol amine, and combinationsthereof.

According to an aspect of the above embodiment, the process furtherincludes convergently applying the modified cellulose solution and thegelation composition onto a treatment site.

According to an aspect of the above embodiment, wherein the modifiedcellulose is an oxidized cellulose.

In one embodiment, the present disclosure provides a process including:forming a modified cellulose solution; and contacting the modifiedcellulose solution with at least one non-solvent to form a plurality ofmodified cellulose particles.

According to an aspect of the above embodiment, the forming of themodified cellulose solution includes: contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose with a salt underthe inert atmosphere to form a modified cellulose solution; andadjusting the modified cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the at least onenon-solvent is selected from the group consisting of alkanes, oilsglycerins, glycols, and combinations thereof.

According to an aspect of the above embodiment, the process furtherincludes shearing the modified solution to form the plurality ofmodified cellulose particles.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the modified celluloseis an oxidized cellulose.

According to an aspect of the above embodiment, the plurality ofmodified cellulose particles include oxidized cellulose having a degreeof oxidation from about 80% to about 120% of a degree of oxidation ofpredissolved oxidized cellulose.

According to an aspect of the above embodiment, the plurality ofmodified cellulose particles include oxidized cellulose having amolecular weight from about 80% to about 120% of the molecular weight ofpredissolved oxidized cellulose.

In one embodiment, the present disclosure provides a process including:forming an oxidized cellulose solution; and contacting the oxidizedcellulose solution with at least one non-solvent to form a plurality ofoxidized cellulose particles having a degree of oxidation from about 80%to about 120% of the degree of oxidation of predissolved oxidizedcellulose.

According to an aspect of the above embodiment, the forming of theoxidized cellulose solution includes: contacting an oxidized cellulosewith a solvent under an inert atmosphere to form a swelled oxidizedcellulose, the oxidized cellulose having a degree of oxidation of fromabout 0.2 to about 1.0; adjusting the swelled oxidized cellulose to afirst temperature; contacting the swelled oxidized cellulose with a saltunder the inert atmosphere to form an oxidized cellulose solution; andadjusting the oxidized cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the process furtherincludes shearing the oxidized solution to form the plurality ofoxidized cellulose particles.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the at least onenon-solvent is selected from the group consisting of alkanes, oilsglycerins, glycols, and combinations thereof.

In one embodiment, the present disclosure provides a process including:forming a modified cellulose solution; and contacting the modifiedcellulose solution with at least one non-solvent to form a plurality ofmodified cellulose particles having a molecular weight from about 80% toabout 100% of the molecular weight of predissolved modified cellulose.

According to an aspect of the above embodiment, the forming of themodified cellulose solution includes: contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose with a salt underthe inert atmosphere to form a modified cellulose solution; andadjusting the modified cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the process furtherincludes shearing the modified solution to form the plurality ofmodified cellulose particles.

According to an aspect of the above embodiment, the at least onenon-solvent is selected from the group consisting of alkanes, oilsglycerins, glycols, and combinations thereof.

In one embodiment, the present disclosure provides a process for forminga composition including: forming a modified cellulose solution; forminga precipitating composition; and contacting the modified cellulosesolution and the precipitating composition at a treatment site therebyprecipitating modified cellulose from the modified cellulose solutionand forming a gel.

According to an aspect of the above embodiment, the formation of themodified cellulose solution includes: contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose with a salt underthe inert atmosphere to form a modified cellulose solution; andadjusting the modified cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the precipitatingcomposition is selected from the group consisting of water, saline,phosphate buffered saline, and combinations thereof.

According to an aspect of the above embodiment, the precipitatingcomposition is an aqueous solution of carboxymethylcellulose, whereinthe carboxymethylcellulose is present from about 0.5% by weight of thesolution to about 5% by weight of the solution.

According to an aspect of the above embodiment, the precipitatingcomposition is a solution of an acrylic polymer based on at least one ofmethyl methacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate,glyceryl acrylate, glyceryl methacrylate, acrylic acid, methacrylicacid, acrylamide, or methacrylamide, and combinations thereof.

According to an aspect of the above embodiment, the precipitationcomposition solution includes a solvent selected from the groupconsisting of acetone, ethyl acetate, dimethyl ether, and combinationsthereof.

According to an aspect of the above embodiment, the process furtherincludes convergently applying the modified cellulose solution and theprecipitating composition onto a treatment site.

In one embodiment, the present disclosure provides a process for forminga composition including: forming a modified cellulose solution; forminga cross-linkable composition covalently cross-linkable with the modifiedcellulose solution; and contacting the modified cellulose solution andthe composition at a treatment site thereby forming a cross-linked gel.

According to an aspect of the above embodiment, the cross-linkablecomposition includes a Schiff-base compound selected from the groupconsisting of amoxicillin, cephalexin, and combinations thereof.

According to an aspect of the above embodiment, cross-linkablecomposition includes trilysine, albumin, polyethylene glycol amine, andcombinations thereof.

According to an aspect of the above embodiment, the cross-linkablecomposition is an aqueous solution.

According to an aspect of the above embodiment, the process furtherincludes convergently applying the modified cellulose solution and thecross-linkable composition onto a treatment site.

In one embodiment, the present disclosure provides a process for forminga composition including: forming a modified cellulose solution; forminga gelation composition; and contacting the modified cellulose solutionand the composition at a treatment site thereby forming a gel.

According to an aspect of the above embodiment, the formation of themodified cellulose solution includes: contacting a modified cellulosewith a solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose with a salt underthe inert atmosphere to form a modified cellulose solution; andadjusting the modified cellulose solution to a second temperature thatis lower than the first temperature.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the gelation compositionis an aqueous solution of chitosan having a pH from about 2.0 to about6.0.

According to an aspect of the above embodiment, the modified cellulosesolution has a pH from about 8.0 to about 9.5.

According to an aspect of the above embodiment, the gelation compositionis an aqueous solution of at least one multivalent cation.

According to an aspect of the above embodiment, the at least onemultivalent cation is selected from the group consisting of cations ofcalcium, barium, zinc, magnesium, chromium, platinum, and iron.

According to an aspect of the above embodiment, the gelation compositionis selected from the group consisting of water, saline, phosphatebuffered saline, and combinations thereof.

According to an aspect of the above embodiment, the gelation compositionis an aqueous solution of carboxymethylcellulose, wherein thecarboxymethylcellulose is present from about 0.5% by weight of thesolution to about 5% by weight of the solution.

According to an aspect of the above embodiment, the gelation compositionis a solution of an acrylic polymer based on at least one of methylmethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, glycerylacrylate, glyceryl methacrylate, acrylic acid, methacrylic acid,acrylamide, or methacrylamide, and combinations thereof.

According to an aspect of the above embodiment, the solution includes asolvent selected from the group consisting of acetone, ethyl acetate,dimethyl ether, and combinations thereof.

According to an aspect of the above embodiment, the gelation compositionincludes a Schiff-base compound selected from the group consisting ofamoxicillin, cephalexin, and combinations thereof.

According to an aspect of the above embodiment, the gelation compositionincludes trilysine, albumin, polyethylene glycol amine, and combinationsthereof.

According to an aspect of the above embodiment, the process furtherincludes convergently applying the modified cellulose solution and thegelation composition onto a treatment site.

In one embodiment, the present disclosure provides a process including:forming a modified cellulose solution; and contacting the dissolvedmodified cellulose with at least one neutralizing agent to form aplurality of modified cellulose particles.

According to an aspect of the above embodiment, forming of the modifiedcellulose solution includes: contacting a modified cellulose with asolvent under an inert atmosphere to form a swelled modified cellulose;adjusting the swelled modified cellulose mixture to a first temperature;contacting the swelled modified cellulose with a salt under the inertatmosphere to form a modified cellulose solution; and adjusting themodified cellulose solution to a second temperature that is lower thanthe first temperature.

According to an aspect of the above embodiment, the at least oneneutralizing agent is selected from the group consisting of ammonia,ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodiumcarbonate, sodium bicarbonate, lithium hydroxide, potassium carbonate,potassium bicarbonate, and combinations thereof.

According to an aspect of the above embodiment, the process furtherincludes shearing the dissolved modified solution to form the pluralityof modified cellulose particles.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the modified celluloseis an oxidized cellulose and the plurality of modified celluloseparticles include oxidized cellulose having a degree of oxidation fromabout 80% to about 120% of a degree of oxidation of predissolvedoxidized cellulose.

According to an aspect of the above embodiment, the plurality ofmodified cellulose particles include oxidized cellulose having amolecular weight from about 80% to about 120% of the molecular weight ofpredissolved oxidized cellulose.

In one embodiment, the present disclosure provides a process including:forming an oxidized cellulose solution; and contacting the dissolvedoxidized cellulose with at least one neutralizing agent to form aplurality of oxidized cellulose particles having a degree of oxidationfrom about 80% to about 120% of the degree of oxidation of predissolvedoxidized cellulose.

According to an aspect of the above embodiment, the forming of theoxidized cellulose solution includes: contacting an oxidized cellulosewith a solvent under an inert atmosphere to form a swelled oxidizedcellulose having a degree of oxidation of from about 0.2 to about 1.0;adjusting the swelled oxidized cellulose to a first temperature;contacting the swelled oxidized cellulose with a salt under the inertatmosphere to form an oxidized cellulose solution; and adjusting theoxidized cellulose solution to a second temperature that is lower thanthe first temperature.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the process furtherincludes shearing the dissolved oxidized solution to form the pluralityof oxidized cellulose particles.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the at least oneneutralizing agent is selected from the group consisting of ammonia,ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodiumcarbonate, sodium bicarbonate, lithium hydroxide, potassium carbonate,potassium bicarbonate, and combinations thereof.

In one embodiment, the present disclosure provides a process including:forming a modified cellulose solution; and contacting the dissolvedmodified cellulose with at least one neutralizing agent to form aplurality of modified cellulose particles having a molecular weight fromabout 80% to about 100% of the molecular weight of predissolved modifiedcellulose.

According to an aspect of the above embodiment, forming of the modifiedcellulose solution includes: contacting a modified cellulose with asolvent under an inert atmosphere to form a swelled modified cellulose;adjusting the swelled modified cellulose mixture to a first temperature;contacting the swelled modified cellulose with a salt under the inertatmosphere to form a modified cellulose solution; and adjusting themodified cellulose solution to a second temperature that is lower thanthe first temperature.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the process furtherincludes shearing the dissolved modified solution to form the pluralityof modified cellulose particles.

According to an aspect of the above embodiment, the at least oneneutralizing agent is selected from the group consisting of ammonia,ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodiumcarbonate, sodium bicarbonate, lithium hydroxide, potassium carbonate,potassium bicarbonate, and combinations thereof.

In one embodiment, the present disclosure provides a process for formingmicrospheres including: contacting a solvent with a modified celluloseto form a solution; contacting the modified cellulose solution with atleast one bioactive agent to form a discontinuous phase liquid;contacting the discontinuous phase liquid with a continuous phase liquidto form an emulsion; and contacting the emulsion with a third phaseliquid to extract the solvent from the emulsion, thereby forming aplurality of modified cellulose microspheres.

According to an aspect of the above embodiment, the bioactive agent isselected from the group consisting of a hydrophilic bioactive agent, aprotein therapeutic, a biologic, and combinations thereof.

According to an aspect of the above embodiment, the third phase liquidis miscible with the continuous phase liquid and the discontinuous phaseliquid.

According to an aspect of the above embodiment, the third phase liquidis selected from the group consisting of isopropyl myristate, hexane,triglycerides and combinations thereof.

According to an aspect of the above embodiment, the third phase liquidis present in an amount from about 130% by volume to about 170% byvolume of the continuous phase liquid.

According to an aspect of the above embodiment, the formation of themodified cellulose solution includes: contacting a modified cellulosewith the solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose after adjustingthe first temperature with a salt under the inert atmosphere to form amodified cellulose solution; and adjusting the modified cellulosesolution to a second temperature from about 90° C. to about 120° C.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the continuous phase isselected from the group consisting of plant-based oils, petroleum-basedoils, silicone-based oils, and combinations thereof.

According to an aspect of the above embodiment, the process furtherincludes: contacting the plurality of modified cellulose microsphereswith a solution of a biodegradable polymer and an aqueous solution toform an emulsion; and extracting a plurality of biodegradable polymermicrospheres encapsulating the plurality of modified cellulosemicrospheres.

According to an aspect of the above embodiment, the biodegradablepolymer is an aliphatic polyester.

According to an aspect of the above embodiment, the aqueous solutionincludes at least one emulsifier and water.

According to an aspect of the above embodiment, the at least onebioactive agent is hydrophilic.

In one embodiment, the present disclosure provides a microsphereincluding: modified cellulose; and at least one bioactive agent.

According to an aspect of the above embodiment, the bioactive agent isselected from the group consisting of a hydrophilic bioactive agent, aprotein therapeutic, a biologic, and combinations thereof.

According to an aspect of the above embodiment, the microsphere isformed by: contacting a modified cellulose solution including a solventwith the at least one bioactive agent to form a discontinuous phaseliquid; contacting the discontinuous phase liquid with a continuousphase liquid to form an emulsion; and contacting the emulsion with athird phase liquid to extract the solvent from the emulsion therebyforming a plurality of microspheres.

According to an aspect of the above embodiment, the third phase liquidis miscible with the continuous composition and the discontinuouscomposition.

According to an aspect of the above embodiment, the third phase liquidis selected from the group consisting of isopropyl myristate, hexane,triglycerides and combinations thereof.

According to an aspect of the above embodiment, the third phase liquidis present in an amount from about 130% by volume to about 170% byvolume of the continuous phase liquid.

According to an aspect of the above embodiment, the formation of themodified cellulose solution includes: contacting a modified cellulosewith the solvent under an inert atmosphere to form a swelled modifiedcellulose; adjusting the swelled modified cellulose mixture to a firsttemperature; contacting the swelled modified cellulose after adjustingthe first temperature with a salt under the inert atmosphere to form amodified cellulose solution; and adjusting the modified cellulosesolution to a second temperature from about 90° C. to about 120° C.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the continuous phase isselected from the group consisting of plant-based oils, petroleum-basedoils, silicone-based oils, and combinations thereof.

In one embodiment, the present disclosure provides a process for formingmicrospheres including: forming a first plurality microspheres includingat least one bioactive agent and modified cellulose; contacting thefirst plurality of microspheres with a solution of a biodegradablepolymer to form a discontinuous phase liquid; contacting thediscontinuous phase liquid with a continuous phase liquid to form anemulsion; and extracting a second plurality of microspheres from theemulsion, the second plurality of microspheres including the firstplurality of microspheres.

According to an aspect of the above embodiment, the at least onebioactive agent is selected from the group consisting of a hydrophilicbioactive agent, a protein therapeutic, a biologic, and combinationsthereof.

According to an aspect of the above embodiment, the biodegradablepolymer is an aliphatic polyester.

According to an aspect of the above embodiment, the aliphatic polyesteris selected from the group consisting of polylactide,polylactide-co-glycolide, polylactide-polycaprolactone, and combinationsthereof.

According to an aspect of the above embodiment, the continuous phaseliquid includes at least one emulsifier and water.

According to an aspect of the above embodiment, the method furtherincluding: contacting the second plurality of microspheres with amodified cellulose solution to form a second discontinuous phase liquid;contacting the second discontinuous phase liquid with a secondcontinuous phase liquid to form a second emulsion; and extracting athird plurality of microspheres from the second emulsion, the thirdplurality of microspheres including the second plurality ofmicrospheres.

In one embodiment, the present disclosure provides a process for formingmicrospheres including: forming a first plurality microspheres includinga biodegradable polymer; contacting the first plurality of microsphereswith a modified cellulose solution to form a discontinuous phase liquid;contacting the discontinuous phase liquid with a continuous phase liquidto form an emulsion; and extracting a second plurality of microspheresfrom the emulsion, the second plurality of microspheres including thefirst plurality of microspheres.

According to an aspect of the above embodiment, at least one of thefirst plurality of microspheres or the modified cellulose solutionincludes at least one bioactive agent.

According to an aspect of the above embodiment, the at least onebioactive agent is selected from the group consisting of a hydrophilicbioactive agent, a protein therapeutic, a biologic, and combinationsthereof.

According to an aspect of the above embodiment, the biodegradablepolymer is an aliphatic polyester.

According to an aspect of the above embodiment, the aliphatic polyesteris selected from the group consisting of polylactide,polylactide-co-glycolide, polylactide-polycaprolactone, and combinationsthereof.

According to an aspect of the above embodiment, the process includes:contacting the second plurality of microspheres with a second solutionof a biodegradable polymer to form a second discontinuous phase liquid;contacting the second discontinuous phase liquid with a secondcontinuous phase liquid to form a second emulsion; and extracting athird plurality of microspheres from the second emulsion, the thirdplurality of microspheres including the second plurality ofmicrospheres.

In one embodiment, the present disclosure provides a microsphereincluding a first biodegradable polymer encapsulating at least oneadditional microsphere, the at least one additional microsphereincluding a second biodegradable polymer and at least one bioactiveagent, wherein the first biodegradable polymer and the secondbiodegradable polymer are different and at least one of the firstbiodegradable polymer or the second biodegradable polymer is modifiedcellulose.

According to an aspect of the above embodiment, at least one of thefirst biodegradable polymer or the second biodegradable polymer is analiphatic polyester.

According to an aspect of the above embodiment, aliphatic polyester isselected from the group consisting of polylactide,polylactide-co-glycolide, polylactide-polycaprolactone, and combinationsthereof.

According to an aspect of the above embodiment, the microsphere furtherincludes at least one additional bioactive agent.

According to an aspect of the above embodiment, the at least onebioactive agent and the at least one additional bioactive agent areselected from the group consisting of a hydrophilic bioactive agent, aprotein therapeutic, a biologic, and combinations thereof and the firstbiodegradable polymer is modified cellulose.

According to an aspect of the above embodiment, the first biodegradablepolymer encapsulating the at least one additional microsphere is furtherencapsulated in a third microsphere formed of the second biodegradablepolymer.

According to an aspect of the above embodiment, the first biodegradablepolymer is selected from the group consisting of polylactide,polylactide-co-glycolide, polylactide-polycaprolactone, and combinationsthereof and the second biodegradable polymer is modified cellulose.

According to an aspect of the above embodiment, the first biodegradablepolymer is modified cellulose and the second biodegradable polymer isselected from the group consisting of polylactide,polylactide-co-glycolide, polylactide-polycaprolactone, and combinationsthereof.

In one embodiment, the present disclosure provides a medical deviceincluding at least one of a predictably degrading coating, film, or afiber formed from a modified cellulose solution.

According to an aspect of the above embodiment, the solution is formedby: contacting a modified cellulose with a solvent under an inertatmosphere to form a swelled modified cellulose; adjusting the swelledmodified cellulose mixture to a first temperature; contacting theswelled modified cellulose with a salt under the inert atmosphere toform a modified cellulose solution; and adjusting the modified cellulosesolution to a second temperature.

According to an aspect of the above embodiment, the at least one of thefiber, the coating, or the film are formed by evaporating the solventfrom the solution.

According to an aspect of the above embodiment, the at least one of thefiber, the coating, or the film are formed by depositing the modifiedcellulose solution on a substrate and evaporating the solvent from thesolution.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the modified celluloseis an oxidized cellulose and the at least one of the fiber, the coating,or the film include oxidized cellulose having a degree of oxidation fromabout 80% to about 120% of the degree of oxidation of predissolvedoxidized cellulose.

According to an aspect of the above embodiment, the at least one of thefiber, the coating, or the film include oxidized cellulose having amolecular weight from about 80% to about 120% of the molecular weight ofpredissolved oxidized cellulose.

According to an aspect of the above embodiment, the modified cellulosesolution is contacted with at least one biocompatible plasticizer.

According to an aspect of the above embodiment, the at least onebiocompatible plasticizer is selected from the group consistinglecithin, dibutyl sebacate, citric acid, polyethylene glycol,polypropylene glycol, and combinations thereof.

In one embodiment, the present disclosure provides a process including:contacting a modified cellulose with a solvent under an inert atmosphereto form a swelled modified cellulose; adjusting the swelled modifiedcellulose mixture to a first temperature; contacting the swelledmodified cellulose with a salt under the inert atmosphere to form amodified cellulose solution; adjusting the modified cellulose solutionto a second temperature that is lower than the first temperature; andforming at least one of a fiber, a coating, or a film from the modifiedcellulose solution.

According to an aspect of the above embodiment, the at least one of thefiber, the coating, or the film are formed by evaporating the solventfrom the solution.

According to an aspect of the above embodiment, the first temperature isfrom about 115° C. to about 145° C. and the second temperature is fromabout 90° C. to about 120° C.

According to an aspect of the above embodiment, the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.

According to an aspect of the above embodiment, the salt is selectedfrom the group consisting of lithium halides, sodium halides, potassiumhalides, and combinations thereof.

According to an aspect of the above embodiment, the modified celluloseis an oxidized cellulose and the at least one of the fiber, the coating,or the film include oxidized cellulose having a degree of oxidation fromabout 80% to about 120% of a degree of oxidation of predissolvedoxidized cellulose.

According to an aspect of the above embodiment, the at least one of thefiber, the coating, or the film include oxidized cellulose having amolecular weight from about 80% to about 120% of the molecular weight ofpredissolved oxidized cellulose.

According to an aspect of the above embodiment, the process furtherincludes contacting the modified cellulose solution with at least onebiocompatible plasticizer.

According to an aspect of the above embodiment, the at least onebiocompatible plasticizer is selected from the group consistinglecithin, dibutyl sebacate, citric acid, polyethylene glycol,polypropylene glycol, and combinations thereof.

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments of the present disclosure will be described hereinbelow with reference to the figures wherein:

FIG. 1 is a schematic diagram of a system for dissolving cellulose inaccordance with the present disclosure;

FIG. 2 is a schematic diagram of a doubly-encapsulated microsphere inaccordance with the present disclosure;

FIG. 3 is a schematic diagram of a multi-encapsulated microsphere inaccordance with the present disclosure;

FIG. 4 is a plot of a release profile of a multi-encapsulatedmicrosphere including a plurality of bioactive agents in accordance withthe present disclosure;

FIG. 5 is a plot of a release profile of a multi-encapsulatedmicrosphere including a single bioactive agent in accordance with thepresent disclosure;

FIG. 6 is a graph of a chromatogram of oxidized cellulose dissolved inaccordance with the present disclosure;

FIG. 7 is a graph of a chromatogram of non-modified cellulose dissolvedin accordance with the present disclosure; and

FIGS. 8A-B are scanning electron microscope images of oxidized cellulosemicrospheres in accordance with the present disclosure;

FIGS. 9A-B are scanning electron microscope image of oxidized cellulosemicroparticles including 18% loaded vitamin B-12 in accordance with thepresent disclosure;

FIGS. 10A-B are scanning electron microscope images of oxidizedcellulose microparticles including bupivacaine free base in accordancewith the present disclosure;

FIGS. 11A-B are scanning electron microscope images of oxidizedcellulose microspheres including bupivacaine hydrochloride form inaccordance with the present disclosure;

FIG. 12 is an ultraviolet-visible spectroscopy standard calibrationcurve for vitamin B-12 in accordance with the present disclosure;

FIGS. 13A-B are scanning electron microscope image of oxidized cellulosemicroparticles including 30% loaded vitamin B-12 in accordance with thepresent disclosure;

FIGS. 14A-B are scanning electron microscope image of oxidized cellulosemicroparticles including 25% loaded vitamin B-12 in accordance with thepresent disclosure;

FIG. 15 is a light microscope image of cis-diamminedichloroplatinum(II)loaded oxidized cellulose microspheres in accordance with the presentdisclosure;

FIG. 16 is a light microscope image of poly-D,L,-lactide microspheresencapsulating cis-diamminedichloroplatinum(II) loaded oxidized cellulosemicrospheres of FIG. 15 in accordance with the present disclosure;

FIG. 17 is a scanning electron microscope image of a cross-section ofthe microsphere of FIG. 16 in accordance with the present disclosure;

FIG. 18 is a plot of a conductometric titration curve of oxidizedcellulose in accordance with the present disclosure; and

FIG. 19 is a plot of a pH-metric titration curve of oxidized cellulosein accordance with the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure provides a system and method for dissolvingcellulose. In embodiments, the present disclosure provides a processusing a polar aprotic solvent and a salt, which is added in a step-wisemanner to dissolve oxidized or non-modified cellulose. The dissolutionprocess according to the present disclosure minimizes degradation of theoxidized cellulose, by conducting the process in an inert and dryatmosphere, introducing the salt in a specific sequence, heating thesolution at a predetermined temperature and time, and minimizingshearing forces on the solution.

As described herein, cellulose includes natural (e.g., non-modified) ormodified (e.g., treated) celluloses including, but not limited to,oxidized cellulose, alkyl celluloses, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitrocelluloses, combinations thereof, and thelike. Additional examples of suitable modified cellulose derivativesinclude, but are not limited to, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, celluloseacetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose,cellulose triacetate, and cellulose sulfate sodium salt.

As used herein, oxidized cellulose denotes cellulose having at least aportion of hydroxyl groups replaced by carboxyl, aldehyde, and/or ketonegroups by oxidation. Oxidized cellulose may be formed using anytechnique within the purview of those skilled in the art. For example,cellulose may be oxidized by exposing it to an oxidation medium, such asa densified or supercritical fluid including, but not limited to,nitrogen dioxide, carbon dioxide, combinations thereof, and the like. Inembodiments, the oxidation medium may include a combination of densifiedor supercritical fluids, such as nitrogen dioxide dissolved in carbondioxide. The cellulose material may be exposed to the oxidizing mediumfor a period of time of from about 20 minutes to about 24 hours, inembodiments from about 1 hour to about 5 hours, at a temperature fromabout 20° C. to about 60° C., in embodiments from about 30° C. to about45° C., and at a pressure of from about 20 bars to about 250 bars, inembodiments from about 30 bars to about 90 bars. Methods for oxidizingcellulose materials using densified fluids are disclosed, for example,in U.S. Patent Application Publication No. 2008/0194805, the entiredisclosure which is incorporated by reference herein. Other methods forpreparing oxidized cellulose materials are also disclosed, for example,in U.S. Pat. Nos. 3,364,200; 4,626,253; 5,484,913; and 6,500,777, theentire disclosures, of each of which are incorporated by referenceherein.

Turning now to FIG. 1, a system for dissolving cellulose, includingoxidized cellulose, in accordance with the present disclosure isprovided. System 10 includes a reactor vessel 12, which may be athree-neck round-bottom flask. The reactor vessel 12 includes a gasinlet 14 and a gas outlet 16, both of which are coupled to a source ofinert gas (not shown). The reactor vessel 12 may also include any numberof inlets, spigots, and other connectors to provide for convenientaddition of reactants and/or removal of products to or from the vessel12, respectively. Dissolution of the oxidized cellulose may be carriedout either as a continuous process or a batch process.

The dissolution process is performed in an inert, i.e., oxygen free, anddry atmosphere. In embodiments, the reactor vessel 12 may be purged withan inert gas prior to commencing the dissolution process by circulatingan inert gas through the reactor vessel 12 via the inlet 14 and outlet16. The gas may also be circulated through the reactor vessel 12 duringthe dissolution process. Suitable inert gases include, but are notlimited to, nitrogen and noble gases such as helium, neon, argon, andcombinations thereof.

Initially, a solvent is added to the reactor vessel 12 through anysuitable inlet. In embodiments, the solvent for dissolving oxidizedcellulose may be any polar aprotic organic solvent having a boilingpoint from about 175° C. to about 205° C., in embodiments from about180° C. to about 202° C. Suitable solvents include, but are not limitedto, N,N-Dimethylacetamide, N-methyl-2-pyrrolidinone (NMP), andcombinations thereof.

The solvent may also be sparged (e.g., gas bubbled therethrough) by theinert gas to exclude moisture and dissolved oxygen therefrom. Celluloseis then added to the solvent and may be agitated by a mixer 18 to swellthe cellulose. Mixing is performed at a relatively low rate to preventdegradation of the cellulose. The stirring may be from about 100revolutions per minute (rpm) to about 500 rpm, in embodiments from about150 rpm to about 250 rpm. As described above, the reactor vessel 12 maybe a round-bottomed container, which further minimizes the shearingforces imparted on the cellulose by the mixer 18.

The mixture of the solvent and oxidized cellulose may be heated to atemperature from about 115° C. to about 145° C., in embodiments fromabout 120° C. to about 140° C. in further embodiments from about 130° C.to about 135° C. In embodiments, the degree of oxidation of oxidizedcellulose dissolved using the processes in accordance with the presentdisclosure may be from about 0.2 to about 1.0, in embodiments from about0.3 to about 0.9, in further embodiments from about 0.5 to about 0.7. Asused herein, the term “degree of oxidation” refers to a ratio ofcarboxyl groups to hydroxyl groups of the cellulose. The “degree ofoxidation” is also used as an average degree of oxidation of the entirecellulose sample. Without being bound by any particular theory, it isbelieved that the temperature of the mixture of the solvent and oxidizedcellulose depends on the degree of oxidation of the oxidized cellulose.As the degree of oxidation increases, the temperature required to swelloxidized cellulose decreases. Conversely, as the degree of oxidationdecreases, the temperature required to swell oxidized celluloseincreases. Heating of the cellulose during the dissolution process isminimized. Heating of the cellulose may lead to degradation thereof,including destruction of reactive groups of oxidized cellulose anddecrease in molecular weight.

The mixture of the solvent and oxidized cellulose having a degree ofoxidation of about 0.5 or above may be heated to a temperature fromabout 115° C. to about 135° C., in embodiments from about 125° C. toabout 130° C. The mixture of the solvent and oxidized cellulose having adegree of oxidation of from about 0.25 to about 0.5 may be heated to atemperature from about 130° C. to about 145° C., in embodiments fromabout 135° C. to about 140° C.

The solvent initially swells the cellulose due to its relatively highpolarity. Swelling of oxidized cellulose may continue from about 1 hourto about 4 hours, in embodiments from about 1.5 hours to about 2.5hours. After the oxidized cellulose has swelled, the temperature of themixture is reduced. In embodiments, the mixture of oxidized cellulosemay be cooled prior to addition of the salt to a temperature from about90° C. to about 120° C., in embodiments from about 100° C. to about 110°C.

Without being bound by any particular theory, it is believed thatintroduction of the salt into the mixture provides intercalation of thesalt into the cellulose. The swelling of the cellulose with the solventenhances the introduction of the salt into the cellulose, which in turn,affects final dissolution of the cellulose. In embodiments, the salt maybe any alkali halide salt. Suitable salts include, but are not limitedto, lithium halides, such as lithium fluoride, lithium chloride, lithiumbromide, and lithium iodide; sodium halides, such as sodium fluoride,sodium chloride, sodium bromide, and sodium iodide; potassium halides,such as potassium fluoride, potassium chloride, potassium bromide, andpotassium iodide; and any combinations of the foregoing. The salt may bepresent in an amount of from about 0.1% by weight to 3% by weight of theoxidized cellulose, in embodiments from about 0.25% by weight to about2% by weight of the oxidized cellulose. Conventional dissolutionprocesses rely on higher salt concentration to dissolve non-modifiedcellulose, which are unsuitable for dissolving oxidized cellulose. Lowerconcentration of salt prevents or lessens degradation of oxidizedcellulose including destruction of reactive groups of oxidized celluloseand decrease in molecular weight as described above.

Conducting the dissolution process in a step-wise manner, namely,initial swelling of the cellulose in the solvent prior to introductionof the salt, allows for dissolution of the cellulose at lowertemperatures than conventional processes, which usually requiretemperatures above 150° C. The step-wise dissolution process at lowertemperatures also prevents or lessens degradation of oxidized celluloseincluding destruction of reactive groups of oxidized cellulose anddecrease in molecular weight as described above. In embodiments, thedegree of oxidation of the dissolved oxidized cellulose may be fromabout 80% to about 120% of the degree of oxidation of the pre-processed,i.e., undissolved, oxidized cellulose, in embodiments from about 90% toabout 110%. In embodiments, the molecular weight of the dissolvedoxidized cellulose may be from about 80% to about 100% of the molecularweight of the pre-processed, i.e., undissolved, oxidized cellulose, inembodiments from about 90% to about 95%. As used herein, the term“molecular weight” refers to weight average molecular weight (Mw) of thecellulose. This term “molecular weight” is also used as an averagemolecular mass of the entire cellulose sample. Undissolved (e.g., priorto dissolution) oxidized cellulose may have a molecular weight fromabout 50,000 Daltons to about 500,000 Daltons, in embodiments from about100,000 Daltons to about 400,000 Daltons.

If the oxidized cellulose is not fully dissolved, the process maycontinue with stirring and heating at a lower temperature from about 40°C. to about 80° C., in embodiments from about 50° C. to about 60° C.,for a period of time from about 1 hour to about 5 hours, in embodimentsfrom about 2 hours to about 3 hours, until the oxidized cellulose isdissolved. The resulting solution of oxidized cellulose includesoxidized cellulose present at a concentration of from about 5 milligramsper milliliter (mg/mL) to about 25 mg/mL, in embodiments from about 10mg/mL to about 20 mg/mL.

The system of FIG. 1 may also be used to dissolve non-modifiedcellulose. The process for dissolving non-modified cellulose may utilizethe same solvents as described above for dissolving oxidized cellulose.Initially, the non-modified cellulose is swelled in the solvent. Themixture of the solvent and non-modified cellulose may be heated to atemperature from about 135° C. to about 165° C., in embodiments fromabout 145° C. to about 155° C. The solvent initially swells thecellulose due to its relatively high polarity. Swelling of non-modifiedcellulose may continue from about 1 hour to about 4 hours, inembodiments from about 1.5 hours to about 2.5 hours. After thenon-modified cellulose has swelled, the temperature of the mixture isreduced. In embodiments, the mixture of non-modified cellulose may becooled prior to addition of the salt to a temperature from about 140° C.to about 160° C., in embodiments from about 145° C. to about 155° C.

The salt may be present in an amount of from about 0.1% by weight to 10%by weight of the non-modified cellulose, in embodiments from about 0.5%by weight to about 9% by weight of the non-modified cellulose. If thenon-modified cellulose is not fully dissolved, the process may continuewith stirring and heating at a lower temperature, from about 40° C. toabout 80° C., in embodiments from about 50° C. to about 60° C., for aperiod of time from about 12 hours to about 36 hours, in embodimentsfrom about 16 hours to about 24 hours, until the non-modified celluloseis dissolved.

The dissolved oxidized cellulose may then be used to form macro, microor nanoparticles. In the present application, the terms“macroparticles,” “macrospheres,” “macrocapsules,” “microparticles,”“microspheres,” “microcapsules,” “nanoparticles,” “nanospheres,” and“nanocapsules” are used interchangeably and denote any particle havingany regular or irregular shape and size from about 0.001 μm to about 2mm, in embodiments from about 0.01 μm to about 1 mm.

Particle formation may be carried out either in as a continuous processwith the dissolution process (e.g., subjecting the solution to highshearing forces, adding neutralizing agents, and/or adding cations) or abatch process. In embodiments, cellulose particles may be formed bysubjecting the dissolved cellulose to high shearing forces (e.g., in ahigh-shear apparatus such as a mixer, extruder, and the like) in thepresence of a solvent or non-solvent, a neutralizing agent, an aqueoussolution having multivalent cations, and combination thereof.

The term “non-solvent”, as used herein, is used in its broadest senseand includes any substance or mixture of substances in which celluloseis not soluble. Suitable solvents and co-solvents include, but are notlimited to, NMP, DMAc and aqueous solutions, and combinations thereof.Suitable non-solvents include, but are not limited to, alkanes, oilsglycerins, glycols, and combinations thereof. The solvent or non-solventmay be present in an amount of from about 1% by weight to 45% by weightof the cellulose, in embodiments from about 5% by weight to about 30% byweight of the cellulose, in embodiments from about 10% by weight to 20%by weight of the cellulose.

In embodiments, oxidized cellulose particles may be formed by contactingthe dissolved cellulose with an aqueous solution having a neutralizingagent. The dissolved cellulose and the aqueous neutralizing solution mayalso be subjected to high shearing forces. In embodiments, theneutralizing agent may be used to neutralize the pendant carboxyl acidgroups in the cellulose to regulate the final particle size andmorphology, so a neutralizing agent herein may also be referred to as a“basic neutralization agent.” Any suitable basic neutralization reagentmay be used in accordance with the present disclosure. In embodiments,suitable basic neutralization agents may include both inorganic basicagents and organic basic agents. Suitable basic agents may includeammonia, ammonium hydroxide, potassium hydroxide, sodium hydroxide,sodium carbonate, sodium bicarbonate, lithium hydroxide, potassiumcarbonate, potassium bicarbonate, combinations thereof, and the like.Suitable basic agents may also include monocyclic compounds andpolycyclic compounds having at least one nitrogen atom, such as, forexample, secondary amines, which include aziridines, azetidines,piperazines, piperidines, pyridines, bipyridines, terpyridines,dihydropyridines, morpholines, N-alkylmorpholines,1,4-diazabicyclo[2.2.2]octanes, 1,8-diazabicycloundecanes,1,8-diazabicycloundecenes, dimethylated pentylamines, trimethylatedpentylamines, pyrimidines, pyrroles, pyrrolidines, pyrrolidinones,indoles, indolines, indanones, benzindazones, imidazoles,benzimidazoles, imidazolones, imidazolines, oxazoles, isoxazoles,oxazolines, oxadiazoles, thiadiazoles, carbazoles, quinolines,isoquinolines, naphthyridines, triazines, triazoles, tetrazoles,pyrazoles, pyrazolines, and combinations thereof. In embodiments, themonocyclic and polycyclic compounds may be unsubstituted or substitutedat any carbon position on the ring.

The neutralizing agent may be utilized as a solid such as, for example,sodium hydroxide flakes and may be dissolved in water to form an aqueoussolution. The neutralizing agent may be added to the oxidized cellulosesuch that the pH of the solution is from about 5 to about 9, inembodiments from about 6 to about 8. As noted above, the basicneutralization agent may be added to neutralize the cellulose possessingcarboxylic acid groups (e.g., oxidized cellulose). Neutralization of thependant carboxylic acids in the formation of cellulose particles byminimizing inter-particle repulsion from anionic charges of thecarboxylic acid groups. The addition of the basic neutralization agentmay thus raise the pH of an emulsion including a cellulose possessingacid groups to a pH of from about 5 to about 12, in embodiments, fromabout 6 to about 11.

In embodiments, oxidized cellulose particles may be formed by contactingthe dissolved cellulose with an aqueous solution having multivalentcations, including divalent and trivalent cations. The dissolvedcellulose and the cation solution may also be subjected to high shearingforces. In embodiments, cellulose particles may be formed by acontinuous two-phase spray preparation, in which a cation solution isinitially sprayed onto a subtracted followed by spraying of a dissolvedcellulose solution. In further embodiments, a cationic solution may becombined with an oxidized cellulose solution to form cross-linked gelsin situ as described in further detail below.

Suitable cations include, but are not limited to, those of calcium(Ca⁺²), barium (Ba⁺²), zinc (Zn⁺²), magnesium (Mg⁺²), iron (Fe⁺², Fe⁺³),platinum (Pt⁺⁴), chromium (Cr⁺⁶), and combinations thereof. Inembodiments, the cation may be introduced by dissolving a suitable saltof the cation, which include, but are not limited to, halides, sulfates,carbonates, phosphates, nitrates, nitrites, oxides, acetates,combinations thereof, and the like. The cations may be present in anamount of from about 0.01% by weight to 25% by weight of the oxidizedcellulose, in embodiments from about 1% by weight to about 18% by weightof the cellulose, in embodiments from about 2% by weight to 15% byweight of the oxidized cellulose depending upon end use of the oxidizedcellulose solution. Cations act as cross-linking agents by cross-linkingpendant carboxylic groups disposed on oxidized cellulose thereby formingcellulose particles. A dual-compartment spraying device (e.g.,micro-fluidizer) may be used which stores the aqueous cation solutionand the oxidized cellulose solution, which ejects the solutioncontemporaneously thereby mixing the particles and forming particlesthat are deposited on a substrate (e.g., tissue). Applicators for mixingtwo components are disclosed in commonly-owned U.S. Pat. Nos. 7,611,494,8,033,483, 8,152,777 and U.S. Patent Application Publication Nos.2010/0065660 and 2010/0096481, the entire disclosures of all of whichare incorporated by reference herein.

In embodiments, the degree of oxidation of the oxidized celluloseparticles formed from the dissolved oxidized cellulose of the presentdisclosure may be from about 80% to about 120% of the degree ofoxidation of the pre-processed, i.e., undissolved, oxidized cellulose,in embodiments from about 90% to about 110%. In embodiments, themolecular weight of the oxidized cellulose particles may be from about80% to about 100% of the molecular weight of the pre-processed, i.e.,undissolved, oxidized cellulose, in embodiments from about 90% to about95%. Undissolved (e.g., prior to dissolution) oxidized cellulose mayhave a molecular weight from about 50,000 Daltons to about 500,000Daltons, in embodiments from about 100,000 Daltons to about 400,000Daltons.

The dissolved cellulose and/or cellulose particles may be used to formvarious medical devices suitable for a variety of surgical and woundapplications. The medical devices according to the present disclosuremay be any structure suitable for being attached or implanted intotissue, body organs or lumens, including, but not limited to, micro andnano-particles, woven and non-woven fabrics, coatings, patches, films,foams, slit sheets, pledgets, tissue grafts, stents, scaffolds,buttresses, wound dressings, meshes, and/or tissue reinforcements.

In embodiments, as noted above, one or more bioactive agents may beadded to the solvent such that the bioactive agents are incorporatedinto the oxidized cellulose solution, which may then be used to formvarious medical devices. A variety of bioactive agents, including polarand non-polar compounds, are soluble in the solvents described-abovesuitable for forming oxidized cellulose solutions according to thepresent disclosure. In embodiments, the bioactive agent may also beadded after the oxidized cellulose particles have been formed. The terms“bioactive agent” and “active therapeutic agent” (ATA) are usedinterchangeably and in its broadest sense include any substance ormixture of substances that have clinical use. Consequently, bioactiveagents may or may not have pharmacological activity per se, e.g., a dye,or fragrance. Alternatively a bioactive agent could be any agent thatprovides a therapeutic or prophylactic effect, a compound that affectsor participates in tissue growth, cell growth, cell differentiation, ananti-adhesive compound, a compound that may be able to invoke abiological action such as an immune response, or could play any otherrole in one or more biological processes. It is envisioned that thebioactive agent may be applied to the present medical device in anysuitable form of matter, e.g., films, powders, liquids, gels and thelike.

Examples of classes of bioactive agents which may be utilized inaccordance with the present disclosure include anti-adhesives,antimicrobials, analgesics, antipyretics, anesthetics, antiepileptics,antihistamines, anti-inflammatories, cardiovascular drugs, diagnosticagents, sympathomimetics, cholinomimetics, antimuscarinics,antispasmodics, hormones, growth factors, muscle relaxants, adrenergicneuron blockers, antineoplastics, immunogenic agents,immunosuppressants, gastrointestinal drugs, diuretics, steroids, lipids,lipopolysaccharides, polysaccharides, platelet activating drugs,clotting factors and enzymes. It is also intended that combinations ofbioactive agents may be used.

Anti-adhesive agents can be used to prevent adhesions from formingbetween the implantable medical device and the surrounding tissuesopposite the target tissue. In addition, anti-adhesive agents may beused to prevent adhesions from forming between the coated implantablemedical device and the packaging material. Some examples of these agentsinclude, but are not limited to hydrophilic polymers such as poly(vinylpyrrolidone), carboxymethyl cellulose, hyaluronic acid, polyethyleneoxide, poly vinyl alcohols, and combinations thereof.

Suitable antimicrobial agents include triclosan, also known as2,4,4′-trichloro-2′-hydroxydiphenyl ether, chlorhexidine and its salts,including chlorhexidine acetate, chlorhexidine gluconate, chlorhexidinehydrochloride, and chlorhexidine sulfate, silver and its salts,including silver acetate, silver benzoate, silver carbonate, silvercitrate, silver iodate, silver iodide, silver lactate, silver laurate,silver nitrate, silver oxide, silver palmitate, silver protein, andsilver sulfadiazine, polymyxin, tetracycline, aminoglycosides, such astobramycin and gentamicin, rifampicin, bacitracin, neomycin,chloramphenicol, miconazole, quinolones such as oxolinic acid,norfloxacin, nalidixic acid, pefloxacin, enoxacin and ciprofloxacin,penicillins such as oxacillin and pipracil, nonoxynol 9, fusidic acid,cephalosporins, and combinations thereof. In addition, antimicrobialproteins and peptides such as bovine lactoferrin and lactoferricin B maybe included as a bioactive agent in the bioactive coating of the presentdisclosure.

Other bioactive agents include: local anesthetics; non-steroidalantifertility agents; parasympathomimetic agents; psychotherapeuticagents; tranquilizers; decongestants; sedative hypnotics; steroids;sulfonamides; sympathomimetic agents; vaccines; vitamins, such asvitamin A, B-12, C, D, combinations thereof, and the like;antimalarials; anti-migraine agents; anti-parkinson agents such asL-dopa; anti-spasmodics; anticholinergic agents (e.g., oxybutynin);antitussives; bronchodilators; cardiovascular agents such as coronaryvasodilators and nitroglycerin; alkaloids; analgesics; narcotics such ascodeine, dihydrocodeinone, meperidine, morphine and the like;non-narcotics such as salicylates, aspirin, acetaminophen,d-propoxyphene and the like; opioid receptor antagonists, such asnaltrexone and naloxone; anti-cancer agents; anti-convulsants;anti-emetics; antihistamines; anti-inflammatory agents such as hormonalagents, hydrocortisone, prednisolone, prednisone, non-hormonal agents,allopurinol, indomethacin, phenylbutazone and the like; prostaglandinsand cytotoxic drugs; chemotherapeutics, estrogens; antibacterials;antibiotics; anti-fungals; anti-virals; anticoagulants; anticonvulsants;antidepressants; antihistamines; and immunological agents.

Other examples of suitable bioactive agents also include biologics andprotein therapeutics, such as, viruses, bacteria, lipids, amino acids,cells, peptides, polypeptides and proteins, analogs, muteins, and activefragments thereof, such as immunoglobulins, antibodies, cytokines (e.g.,lymphokines, monokines, chemokines), blood clotting factors, hemopoieticfactors, interleukins (IL-2, IL-3, IL-4, IL-6), interferons (β-IFN,α-IFN, and γ-IFN), erythropoietin, nucleases, tumor necrosis factor,colony stimulating factors (e.g., GCSF, GM-CSF, MCSF), insulin,anti-tumor agents and tumor suppressors, blood proteins, fibrin,thrombin, fibrinogen, synthetic thrombin, synthetic fibrin, syntheticfibrinogen, gonadotropins (e.g., FSH, LH, CG, etc.), hormones andhormone analogs (e.g., growth hormone), vaccines (e.g., tumoral,bacterial and viral antigens); somatostatin; antigens; blood coagulationfactors; growth factors (e.g., nerve growth factor, insulin-like growthfactor); bone morphogenic proteins, TGF-B, protein inhibitors, proteinantagonists, and protein agonists; nucleic acids, such as antisensemolecules, DNA, RNA, RNAi; oligonucleotides; polynucleotides; andribozymes.

The present disclosure also provides for compositions and methods offabricating microspheres encapsulating one or more bioactive agentswithin the oxidized cellulose. Suitable bioactive agents are describedin more detail below. Oxidized cellulose microspheres may have abioactive agent loading from about 80% to about 120%, in embodimentsfrom about 90% to about 110%, in further embodiments from about 95% toabout 105%, in additional embodiments from about 98% to about 102%.

Soluble oxidized cellulose, by virtue of being dissolved in a polarsolvent as described above, allows for formation of microspheresincluding hydrophilic bioactive agents encapsulated in the oxidizedcellulose. This may be accomplished by using an oil-in-oil emulsionmethod followed by a solvent extraction step in extraction media. Asused herein the term “emulsion” refers to a mixture of two or moreliquids that are immiscible, in which one liquid form a continuous phaseand the other liquid forms a discontinuous phase. As used herein theterms “discontinuous” and “disperse” phase are used interchangeably andrefer to the compound being dispersed through the continuous phase andmay include the bioactive agent, optional encapsulating polymer and/orcorresponding solvent or solvating agent. As used herein the term“continuous” phase refers to a liquid, such as, oils, that are used toextract the solvent or solvating agent from the discontinuous phase.These liquids are usually immiscible with the solvent employed in thediscontinuous phase. As used herein the terms “thinning agent” and“third” phase are used interchangeably and refer to a liquid thatreduces the viscosity of the continuous phase, is miscible with thecontinuous phase and/or removes residual continuous phase from thesurface of the microsphere. In embodiments, the thinning agent may beimmiscible with the discontinuous phase. As used herein the term“oil-in-oil” emulsion denotes an emulsion in which both the continuousphase and the discontinuous phase are organic liquids.

In forming microspheres of soluble oxidized cellulose by an oil-in-oilsolvent extraction method, one or more hydrophilic bioactive agents maybe added to a solution of oxidized cellulose and are mixed sufficientlyto ensure a uniform suspension or homogeneous solution. Oxidizedcellulose may be present in the solution in an amount from about 0.05%by weight to 45% by weight of the solution, in embodiments, from about5% by weight to about 30% by weight of the solution, in embodiments fromabout 10% by weight to 20% by weight of the solution.

The bioactive agent and oxidized cellulose solution forms thediscontinuous phase, which is added drop-wise to a vessel including aliquid forming a continuous phase. The continuous phase liquid may beany suitable non-polar compound that is immiscible with the polarsolvents used in forming the oxidized cellulose solution. Suitablecontinuous phase liquids include, but are not limited to,petroleum-based oils, such as light, medium or heavy mineral oils (e.g.,mixtures of alkanes having from about 40 carbons to about 60 carbons),plant-based oils, such as cottonseed oil, silicone-based oils, andcombinations thereof. In embodiments, the continuous phase may includetwo or more oils such as, for example, a heavy oil and a light oil, thatcompete for extraction of the discontinuous phase. In embodiments, theheavy oil and the light oil may be present at a ratio of from about 1:5to about 1:1, in embodiments from about 1:3 to about 3:4. Thediscontinuous phase liquid may be present in an amount from about 2% byvolume to about 40% by volume of the continuous phase liquid, inembodiments from about 5% to about 20%.

The vessel possessing the continuous phase may be fitted with a baffle.The vessel may include a mixer with an impeller configured to rotate ata rate of from about 25 rpm to about 60,000 rpm, in embodiments, fromabout 100 rpm to about 15,000 rpm, in further embodiments from about 250rpm to about 5,000 rpm. The stirring may continue from about 5 secondsto about 4 hours, in embodiments, from about 15 seconds to about 1 hour.The rate of rotation may be adjusted to obtain desired particle size.Size of the microsphere particles may be tailored by modulating theduration and the speed of homogenization (e.g., stirring of thediscontinuous and continuous phases), temperature and/or pressure,altering the ratio of continuous to discontinuous phases, the shearrate, and the molecular weight and concentrations of oxidized celluloseand bioactive agents.

Upon completing the transfer of the discontinuous phase solution intothe continuous phase, a third phase liquid may be added to the emulsionto remove the solvent from the discontinuous phase liquid. Suitablethird phase liquids include any compound which is miscible with both thecontinuous and discontinuous phase liquids. The extraction of thesolvent occurs due to the solvent being immiscible in the continuousphase liquid but miscible in the third phase liquid. Suitable thirdphase liquids include isopropyl myristate, hexane, n-heptane,triglycerides and combinations thereof. The third phase liquid may bepresent in an amount from about 130% by volume to about 170% by volumeof the continuous phase liquid, in embodiments from about 140% to about150%.

Removal of the solvent from the continuous phase facilitates formationof microspheres including the bioactive agent encapsulated by theoxidized cellulose. The emulsion may be stirred from about 1 hour toabout 24 hours, in embodiments from about 2 hours to about 5 hours, toaid in the extraction of the polar solvent from the microspheres. Themicrospheres may then be collected via filtration and washed (e.g., withn-heptane) to remove any trace of continuous and discontinuous phaseliquids on the surface of the microspheres. The microspheres may then becollected and transferred into a glass scintillation vial under anitrogen or argon overlay.

The oxidized cellulose microspheres are also suitable for encapsulatinghydrophilic drugs such as bupivacaine HCl as well as viruses, bacteria,amino acids, peptides, proteins, lipids, vaccines, and combinationsthereof since the oil-in-oil emulsion does not react with the waterbarrier of these bioactive agents.

In other embodiments, the oxidized cellulose solution may also be usedto form various types of fibers. In embodiments, fibers may be solid,hollow, porous, and combinations thereof. Fibers may be formed by anysuitable method, including electrospinning, solution casting, extruding,and combinations thereof. The fibers formed from the oxidized cellulosesolutions may be used to form a variety of medical devices. The medicaldevices according to the present disclosure may be any structuresuitable for being attached or implanted into tissue. Suitablestructures formed from the fibers include, for example, films, foams,slit sheets, pledgets, tissue grafts, stents, scaffolds, buttresses,wound dressings, meshes, and/or tissue reinforcements. In embodiments,the fibers may be used to form non-woven meshes or tapes, which may beused as passive hemostats. The non-woven structure of a fibrous meshformed from an oxidized cellulose solution lends itself to use as awound dressing, due to its ability to filter liquids and/or gases.

The oxidized cellulose solution may also be used to form films and/orcoatings. Coatings or films may be formed by depositing the solution byitself or on a substrate solution-casting, dipping, layering,calendaring, spraying, and combinations thereof. The solvent evaporates,thereby forming the film or coating on a substrate. The films may beincorporated onto other medical devices by applying the solution to thesurface of the device, or portion thereof, utilizing any suitable methodwithin the purview of those skilled in the art.

In embodiments, the oxidized cellulose solution may be used to form asprayable delivery vehicle. In further embodiments, the oxidizedcellulose solution may be combined with a second composition that formsa gel or effects precipitation of the oxidized cellulose as described infurther detail below.

The viscosity of the solution for forming fibers, films, and othermedical devices may be adjusted to achieve a desired viscosity. This maybe accomplished by adding one or more plasticizers. Examples of suitableplasticizers include any biocompatible plasticizer, such as lecithin,dibutyl sebacate, citric acid, alcohol esters, polyethylene glycol,polypropylene glycol, and combinations thereof.

Uses for medical devices formed from the dissolved oxidized celluloseinclude closing and healing visceral wall defects and incisions,including incisions due to the removal of tumors, wounds, anastomoses,and fistulae. The medical devices can improve the healing of agastro-intestinal anastomosis and may provide an effective approach forthe management and prevention of fistula. The medical devices may alsoprevent complications of polypectomy (e.g., bleeding and perforation).In embodiments, the medical devices may be reinforced with a mesh (e.g.,formed on a substrate mesh) for the treatment of inguinal hernia and/orincisional hernia.

The rate of in vitro and in vivo biodegradation of medical devicesformed from oxidized cellulose may be regulated by controlling theinitial degree of oxidation of the resultant (e.g., dissolved andprocessed) oxidized cellulose. The greater the degree of oxidation ofthe oxidized cellulose, the faster the rate of biodegradation in vitroand in vivo. The present disclosure provides for processes that minimizethe degradation of the oxidized cellulose during the dissolutionprocess, thereby providing for cellulose having a desired degree ofoxidation. Further, biodegradability of cellulose may be controlled byadjusting the molecular weight and degree of oxidation during thedissolution to provide for predictably degrading oxidized cellulosehaving a predictable degradation profile. Dissolving and processingwithout materially affecting the degree of oxidation allows forpredictable biodegradability of the final products (e.g., medicaldevices). Thus, control of the rate of degradation of the oxidizedcellulose matrix may be accomplished by varying the degree of oxidation,thereby controlling the rate of bioactive agent elution. The degree ofoxidation of the oxidized cellulose may also be adjusted during thedissolution process to achieve a desired degree of oxidation.

Dissolved oxidized cellulose may also be utilized to form in situ gels.Oxidized cellulose solution may be prepared using the methods, e.g.,solvents, conditions, etc., outlined above. The oxidized cellulosesolution may have a pH from about from about 7.0 to about 10.0, inembodiments from about 8.0 to about 9.5. The oxidized cellulose solutionmay be combined with a gelation composition that, upon contacting theoxidized cellulose solution, forms a gel. The gel may be used as anadhesive to seal tissue and/or to provide for delivery of bioactiveagents as described in further detail below.

In embodiments, the oxidized cellulose solution may be combined with acationic material, such as a cationic polysaccharide. In embodiments,the cationic polysaccharide may be chitosan, carboxymethyl chitin, guargum, and combinations, optionally in solution. Chitosan is a naturallinear co-polymer of N-acetyl D-glucosamine (acetylated unit) andD-glucosamine (non-acetylated unit). Chitosan may be produced by partialor full deacetylation of chitin. Chitin may be extracted from naturalsources, e.g., squid, exoskeletons of crustaceans such as shrimp, orvegetable sources such as mushrooms. Chitosan may also be syntheticallyproduced or synthesized by modified microorganisms such as bacteria.

The adhesion of chitosan with other polysaccharides, such as cellulose,includes different kinds of interactions, such as electrostaticinteractions, hydrogen bonds, and hydrophobic interactions, resulting inionic cross-linking with the oxidized cellulose. Chitosan, under certaincircumstances, is a cationic polymer containing NH₃ ⁺ groups. Thepositively charged primary amino groups of chitosan attract anionicgroups of other polymers. Thus, chitosan and anionic polymers are ableto form polyelectrolyte complexes. Polyelectrolyte complex formation mayimprove the mechanical properties of the polymers and lead to newstructures, such as precipitates, films, fibers, and gels.

Adhesion of chitosan with other polymers may also be promoted byenhancing the mechanical properties of the formulation by creatingcovalent bonds between both the components of the adhesive formulation.Chitosan has NH₂ groups which can react covalently with carboxyl groups.Thus, chitosan may be mixed with functionalized polymers having carboxylgroups, such as oxidized cellulose.

The chitosan may have a molecular weight from about 1,000 g/mol to about5,000,000 g/mol, in embodiments from about 5,000 g/mol to about 220,000g/mol. In embodiments, chitosan has a high molecular weight (HMW) offrom about 450,000 g/mol to about 550,000 g/mol. In other embodiments,chitosan has a low molecular weight (LMW) of from about 50,000 g/mol toabout 150,000 g/mol.

A solution of chitosan may be prepared, in embodiments, by dissolvingchitosan in distilled water with a stoichiometric amount of acid, suchas HCl or acetic acid, to ensure the complete protonation of all NH₂groups. The final solution may contain from about 0.5% (w/w) to about 5%(w/w) chitosan, in embodiments from about 2% (w/w) to about 4% (w/w)chitosan. The chitosan solution may have a pH from about from about 1.0to about 7.0, in embodiments from about 2.0 to about 6.0. The lower pHof the chitosan solution allows for suspension of pH sensitive bioactiveagents in one of the solutions, either oxidized cellulose or chitosan,without compromising the bioactivity of the pH sensitive bioactiveagents.

In embodiments, bioactive agents, whose bioactivity is reduced ordestroyed by high pH, such as chemotherapeutic encapsulatedpolypeptides, may be suspended in a chitosan solution and incorporatedinto an in-situ forming gel upon contact with an oxidized cellulosesolution. This gel can be fixed onto a targeted site, such as organs,tissue, etc. and anchor the encapsulated peptide, which then can bereleased. The resulting gel may be either neutral pH upon formation, orthe pH can be adjusted, using the pH of the chitosan solution or theoxidized cellulose solution, to provide a friendly pH environment forthe bioactivity of the peptide to be maintained.

Another suitable composition for gelation with the oxidized cellulosesolution includes an aqueous solution of multi-valent cations, whichforms a gel by ionic cross-linking of the oxidized cellulose andcations. Suitable cations include, but are not limited to, those ofcalcium (Ca⁺²), barium (Ba⁺²), zinc (Zn⁺²), magnesium (Mg⁺²), iron(Fe⁺², Fe⁺³), platinum (Pt⁺⁴), chromium (Cr⁺⁶), and combinationsthereof. In embodiments, the cations may be introduced by dissolving asuitable salt of the cations, which include, but are not limited to,halides, sulfates, carbonates, phosphates, nitrates, nitrites, oxides,combinations thereof, and the like in a suitable solvent such as water,methanol, ethanol, and combinations thereof. The cations may be presentin an amount of from about 0.01% by weight to 25% by weight of thesolution, in embodiments from about 1% by weight to about 18% by weightof the solution, in embodiments from about 2% by weight to 15% by weightof the solution, to achieve a desired mix ratio with the oxidizedcellulose solution. The oxidized cellulose solution and the cationicsolution form a reversible, ionically cross-linked gel. In embodiments,the gel can be made reversible by the addition of anionic solutionsincluding aqueous solutions having a pH of greater than 7.0, such assolutions of urea, ammonia, amino acids such as, lysine and glycine,anionic polysaccharides such as, alginate, dextran, carboxymethylcellulose, and combinations thereof.

A solution of oxidized cellulose may also be contacted with aprecipitation and/or gelation composition that forms a gel by dilutionand/or precipitation of the oxidized cellulose. Precipitation may beaccomplished by contacting the oxidized cellulose solution with acomposition including a solvent or a non-solvent. Suitable gelationcompositions include, but are not limited to, water, saline, phosphatebuffered saline, and combinations thereof. In embodiments, an aqueoussolution of carboxymethyl cellulose may also be used. Carboxymethylcellulose may be present in the solution from about 0.5% by weight orvolume to about 5% by weight or volume, in embodiments, from about 1% byweight or volume to about 2% by weight or volume.

In embodiments, an aqueous solution of any cross-linker having one ormore primary amines including, but not limited to, trilysine, albumin,polyethylene glycol amine, and combinations thereof may be used as aprecipitating gelation composition. In further embodiments, an aqueoussolution of any suitable Schiff-base compound may also be used as aprecipitating gelation composition. As used herein, the term“Schiff-base” compound denotes any compound having a functional groupincluding a carbon-nitrogen double bond with the nitrogen atom connectedto an aryl or an alkyl group having a general formula R₁R₂C═NR₃, whereR₃ and at least one of R₁ or R₂ is an aryl or an alkyl group. SuitableSchiff-base compounds include, but are not limited to, amoxicillin,cephalexin, 2,2-dimethyl benzimidazoline, 2-methyl-2-ethylbenzimidazoline, 2-methyl-2-propyl benzimidazoline, 2-methyl-2-butylbenzimidazoline, 2-methyl-2-hexyl benzimidazoline, 2-methyl-2-decylbenzimidazoline, 2,2-dimethyl-5-methylbenzimidazoline,2-methyl-2-butyl-6-methyl benzimidazoline, 2,2-diethyl benzimidazoline,2,2-diethyl benzimidazoline, 2-ethyl-2-hexyl benzimidazoline,2-methyl-2-isoamyl-5-methyl benzimidazoline, 2,2-dioctylbenzimidazoline, 2,2-didecyl benzimidazoline, 2-propyl-2-pentylbenzimidazoline, 2,2-diethyl-6-ethylbenzimidazoline,2,2-dipropyl-5-isopropylbenzimidazoline,2,2-dipropyl-5-methylbenzimidazoline,2,2-dibutyl-6-methylbenzimidazoline,2,2-dibutyl-6-dodecylbenzimidazoline, 2-methyl-2-propenylbenzimidazoline, 2-ethyl-2-propenyl-5-methylbenzimidazoline,2-methyl-2-butenyl benzimidazoline,2-ethyl-2-butenyl-6-methylbenzimidazoline, 2,2-dihexyl benzimidazoline,2,2-dihexyl-5-methylbenzimidazoline, and combinations thereof.Contacting of Schiff-base compound and/or small molecule cross-linkersolutions with the oxidized cellulose solution results in covalentcross-linking of the oxidized cellulose, which, in turn, produces thegel. In embodiments, the aqueous solution may include CMC as well as theSchiff-base compounds.

In embodiments, a solution of one or more acrylic polymers may also beused to precipitate oxidized cellulose to form gels according to thepresent disclosure. Suitable acrylic polymers include, but are notlimited to, those based on methyl methacrylate, hydroxyethyl acrylate,hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate,acrylic acid, methacrylic acid, acrylamide, methacrylamide, andcombinations thereof. Suitable solvents include acetone, ethyl acetate,dimethyl ether, and combinations thereof.

Upon contact of the oxidized cellulose solution with the precipitatingcomposition, the gel is formed in situ by the dilution of the solventused to form the oxidized cellulose solution and the subsequentprecipitation of the oxidized cellulose. Since the polar solvent of theoxidized cellulose solution is miscible with water and/or organicsolvents described above, oxidized cellulose precipitates out in theform of a gel due to the dilution of the solvent.

In embodiments, the precipitating composition may include a bioactiveagent, which may be suspended in the precipitating composition. Inembodiments, the bioactive agent may be initially suspended in theprecipitating composition as a plurality of microspheres as describedabove. The microspheres may then be re-suspended in either the oxidizedcellulose composition and/or the gelation composition. The resultingoxidized cellulose gel prevents the migration of the microspheres fromthe target site.

As noted above, the gels formed by the solutions of oxidized celluloseand gelation compositions can be used to deliver bioactive agents totissue or the gels may be used to form articles or coatings thereoncontaining bioactive agents. The gels anchor the bioactive agents,microspheres, microparticles, and combinations thereof, to target sites,e.g., organs, tissues, etc. Microspheres and microparticles containingbioactive agents may be formed using the methods described above bysuspending desired bioactive agents in the oxidized cellulose solutionprior to microsphere or microparticle formation. The resulting particlesmay be suspended in the oxidized cellulose solution, which then may becombined with the cationic and/or chitosan solutions. This may beutilized to secure bioactive agents at the desired sites, includingchemotherapeutic agents (e.g., cis-diamminedichloroplatinum(II)) attumor excision sites, to provide for sustained release ofchemotherapeutic agents from the gel and/or the microparticles securedthereby.

The gelation compositions and/or oxidized cellulose solution may be in aliquid form and placed in a syringe or any other suitable deliveryvehicle, such as a sprayer, for immediate or later use. The solutionsmay be placed in delivery vehicles of different volumes so as to reach aspecific ratio of each component.

The solutions may be applied convergently to a desired tissue site toform a gel thereon. As used herein, the term “convergently” denotes atleast partial overlap of the compositions being applied to the substrate(e.g., tissue, medical device, etc.) either during the applicationprocess (e.g., mid-stream) or on a surface of the substrate.

The solutions used to form the gel may also be directly coated on asubstrate, such as a mesh. The substrate may be prepared by soaking itin the desired solutions and drying (e.g., in an oven or in a laminarflow hood). In embodiments, the process may be repeated several times toensure a proper coating displaying the required adhesive properties forthe selected indication of use, e.g., fixation of extraperitoneal orretroperitoneal meshes, skin flap closure, etc.

The ratio of each component may be adjusted to provide a desiredformulation. Each formulation is characterized by its mix ratio (MR). Asused herein, the term “mix ratio” means the amount of the compoundand/or reactive groups responsible for gelation (e.g., free amine groupsof chitosan and/or amount of cations) versus the amount of free carboxylgroups present on the oxidized cellulose. The mix ratio may be at leastabout 1, in embodiments from about 1 to about 40, in further embodimentsfrom about 10 to about 30. In embodiments, each component of the gel maybe diluted with a buffer prior to use for pH adjustment.

The present disclosure also provides for compositions and methods offabricating microspheres having additional microspheres thereinencapsulating one or more APIs or bioactive agents. FIG. 2 shows amicrosphere 20 having one or more microspheres 22 encapsulated therein.As used herein, “multi-encapsulated microspheres” denote theencapsulation of one or more smaller microspheres 22, e.g., particles,spheres, capsules, and combinations thereof in a single largermicrosphere 20. In embodiments, multi-encapsulated microspheres mayencapsulate one or more bioactive agents at same or different loadinglevels.

In a so-called “primary encapsulation,” soluble oxidized cellulose maybe used to encapsulate a bioactive agent, a water-soluble compound, awater-sensitive chemotherapeutic agent and/or active pharmaceuticalingredient, thereby forming oxidized cellulose microspheres, e.g.,microspheres 22, as described above. Primary encapsulation with solubleoxidized cellulose may be carried out using emulsion-based solventevaporation and/or extraction methods including, but not limited to,single-emulsion methods such as oil-in-water (o/w) and water-in-oil(w/o), double-emulsion methods such as water-in-oil-in-water (w/o/w) andsolid-in-oil-in-water (s/o/w), and non-emulsion based methods, such asfluidized-bed, spray-drying, and casting/grinding methods. The primaryoxidized cellulose microspheres may then be further encapsulated inanother biodegradable polymer, other than oxidized cellulose, in aso-called “secondary encapsulation” forming the microsphere 20encapsulating the microspheres 22.

As used herein, the term “biodegradable” in reference to a materialshall refer to the property of the material being able to be harmlesslyabsorbed by the body. In the present application, the terms“biodegradable,” “bioresorbable,” “bioerodable,” and “bioabsorbable” areused interchangeably and are intended to mean the characteristicaccording to which a material decomposes, or loses structural integrityunder body conditions (e.g., enzymatic degradation or hydrolysis) or arebroken down (physically or chemically) under physiologic conditions inthe body, such that the degradation products are excretable orabsorbable by the body after a given period of time. The time period mayvary, from about one hour to about several months or more, depending onthe chemical nature of the material. In embodiments, the material maynot be completely absorbed, provided the non-absorbed material poses nohealth risks and is biocompatible.

Oxidized cellulose microspheres may be formed using oil-in-oilemulsification processes described above. The oxidized cellulosemicrospheres may then be further micro-encapsulated by usingemulsion-based solvent evaporation methods, in which the oxidizedcellulose microspheres are suspended in a solution of a biodegradablepolymer or cross-linked and further encapsulated in another oxidizedcellulose microencapsulation process. The solution may include anysuitable biodegradable polymer, a solvent, and an optional emulsifierand/or a surfactant. In embodiments, additional bioactive agents may beadded to the biodegradable polymer solution, which may be the same ordifferent from the bioactive agent included in the oxidized cellulosemicrospheres. In further embodiments, some rounds of encapsulation mayinclude no bioactive agents based on the desired use and/or performancecharacteristics of multi-encapsulated microspheres (e.g., alteredrelease rate).

Suitable biodegradable polymers used to form microspheres according tothe present disclosure include, but are not limited to, aliphaticpolyesters, polyamides, polyamines, polyalkylene oxalates,poly(anhydrides), polyamidoesters, copoly(ether-esters),poly(carbonates) including tyrosine derived carbonates,poly(hydroxyalkanoates) such as poly(hydroxybutyric acid),poly(hydroxyvaleric acid), and poly(hydroxybutyrate), polyimidecarbonates, poly(imino carbonates) such as such as poly (bisphenolA-iminocarbonate and the like), polyorthoesters, polyoxaesters includingthose containing amine groups, polyphosphazenes, poly (propylenefumarates), polyurethanes, polymer drugs such as polydiflunisol,polyaspirin, and protein therapeutics, biologically modified (e.g.,protein, peptide) bioabsorbable polymers, and copolymers, blockcopolymers, homopolymers, blends, and combinations thereof.

More specifically, aliphatic polyesters include, but are not limited to,polylactide, polylactide-co-glycolide, polylactide-polycaprolactone,homopolymers and copolymers of lactide (including lactic acid, D-,L- andmeso lactide), glycolide (including glycolic acid),epsilon-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylenecarbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylenecarbonate, Δ-valerolactone, β-butyrolactone, γ-butyrolactone,ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one(including its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14-dione),1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomorpholine,pivalolactone, α, α diethylpropiolactone, ethylene carbonate, ethyleneoxalate, 3-methyl-1,4-dioxane-2,5-dione,3,3-diethyl-1,4-dioxan-2,5-dione, 6,8-dioxabicycloctane-7-one, andpolymer blends and copolymers thereof.

Suitable solvents for forming the biodegradable polymer solution of thediscontinuous phase for secondary encapsulation include, but are notlimited to, ethyl acetate, methylene chloride, perchloroethane,trichloroethylene, hexafluoroisopropanol (HFIP), chloroform,tetrahydrofuran, dimethyl formamide, as well as those pharmaceuticalsolvents listed in the ICH Q3C (International Conference onHarmonization—residual solvents used in pharmaceutical processing) andcombinations thereof.

The emulsifier may be present in an amount from about 0.01% by weightand/or volume to about 25% by weight and/or volume of the solvent, inembodiments from about 0.1% by weight and/or volume to about 10% byweight and/or volume of the solvent, in further embodiments from about0.5% by weight and/or volume to about 5% by weight and/or volume of thesolvent. For oil-in-oil processes, the use of an emulsifier is optional.Suitable emulsifiers include, but are not limited to, water-solublepolymers, such as polyvinyl alcohol (“PVA”), polyvinyl pyrrolidone(PVP), polyethylene glycol (PEG), polypropylene glycol (PPG),PLURONICS™, TWEENS™, polysaccharides, phospholipids, and combinationsthereof.

The continuous phase for the secondary encapsulation may also include asurfactant to stabilize the microspheres and adjust the bioactive agentloading efficiency. One, two, or more surfactants may be utilized.Examples surfactants that can be utilized include, for example,polyacrylic acid, methalose, methyl cellulose, ethyl cellulose, propylcellulose, hydroxy ethyl cellulose, carboxy methyl cellulose,polyoxyethylene cetyl ether, polyoxyethylene lauryl ether,polyoxyethylene octyl ether, polyoxyethylene octylphenyl ether,polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate,polyoxyethylene stearyl ether, polyoxyethylene nonylphenyl ether,dialkylphenoxy poly(ethyleneoxy) ethanol, polyoxamers, combinationsthereof, and the like.

Secondary encapsulation of oxidized cellulose microspheres may includecross-linking the microspheres to stabilize subsequent incapsulation andthen forming a suspension of the microspheres in the biodegradablepolymer solution described above. Oxidized cellulose microspheres may becross-linked using any of the cationic species described above. Thesuspension may then be vortexed or intimately stirred to form anemulsion. In embodiments, the oxidized cellulose microspheres may beimmediately suspended in the biodegradable polymer solution withoutcross-linking.

Emulsion-based solvent evaporation may be accomplished by stirring thesuspension or emulsion at a rate from about 25 rpm to about 60,000 rpm,in embodiments, from about 100 rpm to about 15,000 rpm, in furtherembodiments from about 250 rpm to about 5,000 rpm. The emulsion may bestirred for a period of time from about 5 seconds to about 4 hours, inembodiments, from about 15 seconds to about 1 hour. Stirring may also beused to remove the discontinuous phase solvent from the emulsion,retaining the doubly-encased microspheres.

For the second round of encapsulation, the solvent may be evaporatedand/or extracted. After the solvent is evaporated and/or extracted, theemulsion retains the microspheres formed from the biodegradable polymerencapsulating the oxidized cellulose microspheres. The emulsion alsoincludes free unencapsulated oxidized cellulose microspheres that aresuspended in the emulsion. The size of the doubly-encased ormulti-encased microspheres may be from about 0.001 μm to about 2 mm, inembodiments the size of the microspheres may be from about 0.01 μm toabout 1 mm, in further embodiments the size of the microspheres may befrom about 0.1 μm to about 500 μm. Size of the microspheres may betailored by modulating the duration and the speed of stirring,temperature and/or pressure, altering the ratio of continuous todiscontinuous phases, the shear rate created during stirring, and themolecular weight and concentrations of biodegradable polymers,emulsifiers, and surfactants, and other variables within purview of aperson skilled in the art.

The primary encapsulation by the oxidized cellulose protects thebioactive agent from organic solvents used in any subsequentencapsulation. Oxidized cellulosed may be used to encapsulate bothhydrophilic and hydrophobic bioactive agents. While hydrophobicbioactive agents can also be encapsulated using emulsion methodsincluding other biodegradable polymers, encapsulation of hydrophilicbioactive agents is particularly facilitated by dissolved oxidizedcellulose.

Soluble oxidized cellulose, by virtue of being dissolved in a polarsolvent as described above, allows for formation of microspheresincluding hydrophilic and/or hydrophobic bioactive agents encapsulatedin the oxidized cellulose whereas other biodegradable polymers can beused to encapsulate hydrophobic bioactive agents. Using oxidizedcellulose for the first round of microencapsulation is beneficial sinceit does not dissolve in most polar or non-polar solvents, with theexception of solvents listed above with respect to dissolution ofoxidized cellulose, thus eliminating the risk of microsphere dissolutionduring the second round of encapsulation. This allows formicroencapsulation of both hydrophobic and hydrophilic bioactive agents,which can then be encapsulated into another microsphere.

In embodiments, the first layer of any microspheres may be formed usinga biodegradable polymer other than oxidized cellulose usingabove-described encapsulation methods, which can then be furtherencapsulated in oxidized cellulose microspheres. Primary encapsulationof bioactive agents using biodegradable polymers may be carried outusing emulsion-based solvent evaporation methods including, but notlimited to, single-emulsion methods such as oil-in-water (o/w) andwater-in-oil (w/o), double-emulsion methods such aswater-in-oil-in-water (w/o/w) and solid-in-oil-in-water (s/o/w), andnon-emulsion based methods, such as fluidized-bed, spray-drying, andcasting/grinding methods.

Where a bioactive agent is first encapsulated in a biodegradablepolymer, the bioactive agent may be dissolved in a solution to form adiscontinuous phase. Suitable solvents for dissolving bioactive agentsinclude water, saline and alcohols, examples of which include methanol,ethanol, combinations thereof, and the like. Biodegradable polymer mayalso be dissolved to form a discontinuous phase using the solventsdescribed above. Homogenization may be used for discontinuous phases ifparticle size reduction in the loading of the microsphere is desired.Homogenization may be carried by any suitable methods within the purviewof one skilled in the art including, but not limited to, stirring,grinding, thermal energy, ultrasound energy, combinations thereof, andthe like.

Emulsion-based solvent evaporation may be accomplished by stirring thesuspension or emulsion at a rate from about 25 rpm to about 60,000 rpm,in embodiments, from about 100 rpm to about 15,000 rpm, in furtherembodiments from about 250 rpm to about 5,000 rpm. The emulsion may bestirred for a period of time from about 5 seconds to about 4 hours, inembodiments, from about 15 seconds to about 1 hour. Stirring may also beused to remove the discontinuous phase solvent from the emulsion,retaining the doubly-encased microspheres.

After the solvent is evaporated, the emulsion retains the microsphereparticles formed from the biodegradable polymer encapsulating thebioactive agent. The emulsion also includes free unencapsulated portionof the bioactive agent that is suspended in the emulsion. The size ofthe microsphere particles may be from about 0.001 μm to about 2 mm, inembodiments the size of the microspheres may be from about 0.01 μm toabout 1 mm, in further embodiments the size of the microspheres may befrom about 0.1 μm to about 500 μm. Size of the microspheres may betailored by modulating the duration and the speed of stirring,temperature and/or pressure, altering the ratio of continuous todiscontinuous phases, the shear rate created during stirring, and themolecular weight and concentrations of biodegradable polymers,emulsifiers, and surfactants, and other variables within purview of aperson skilled in the art.

The microspheres formed from the biodegradable polymers other thanoxidized cellulose may then be suspended in a solution of oxidizedcellulose, which is formed according to the processes described above.In forming microspheres of soluble oxidized cellulose by asolid-in-oil-in-oil solvent extraction method, the biodegradable polymermicrospheres may be added to a solution of oxidized cellulose and aremixed sufficiently to ensure a uniform suspension. Oxidized cellulosemay be present in the solution in an amount from about 1% by weight to45% by weight of the solution, in embodiments from about 5% by weight toabout 30% by weight of the solution, in embodiments from about 10% byweight to 20% by weight of the solution. In embodiments, additionalbioactive agents may be added to the oxidized cellulose solution whichmay be the same or different from the bioactive agents of thebiodegradable polymer microspheres (e.g., hydrophilic vs hydrophobicbioactive agents).

The microspheres, the oxidized cellulose solution, and additionalbioactive agents, if any, form the discontinuous phase, which is addeddrop-wise to a vessel including a liquid forming a continuous phase. Thecontinuous phase liquid may be any suitable non-polar compound that isimmiscible with the polar solvents used in forming the oxidizedcellulose solution. Suitable continuous phase liquids include, but arenot limited to, light, medium or heavy mineral oil (e.g., mixtures ofalkanes having from about 40 carbons to about 60 carbons), cottonseedoil, and combinations thereof. Additional continuous phase may be addedduring emulsification. The discontinuous phase liquid may be present inan amount from about 2% by volume to about 40% by volume of thecontinuous phase liquid, in embodiments from about 5% to about 20%.

Emulsion-based solvent evaporation may be accomplished by stirring thesuspension or emulsion at a rate from about 25 rpm to about 60,000 rpm,in embodiments, from about 100 rpm to about 15,000 rpm, in furtherembodiments from about 250 rpm to about 5,000 rpm. The emulsion may bestirred for a period of time from about 5 seconds to about 4 hours, inembodiments, from about 15 seconds to about 1 hour. Stirring may also beused to remove the discontinuous phase solvent from the emulsion,retaining the doubly-encased microspheres.

Upon completing the transfer of the discontinuous phase solution intothe continuous phase, a third phase liquid may be added to the emulsionto remove or extract the solvent from the discontinuous phase liquid.Suitable third phase liquids include any compound which is miscible withthe continuous and may be miscible with discontinuous phase solvent. Theextraction of the solvent occurs due to the solvent being immiscible inthe continuous phase liquid but miscible in the third phase liquid.Suitable third phase liquids include isopropyl myristate, hexane,triglycerides and combinations thereof. The third phase liquid may bepresent in an amount from about 130% by volume to about 170% by volumeof the continuous phase liquid, in embodiments from about 140% to about150%.

Extraction of the solvent from the discontinuous phase facilitatesformation of doubly-encased microspheres including the bioactive agentencapsulated by a biodegradable polymer, other than oxidized celluloseand then further encapsulated by the oxidized cellulose. The emulsionmay be stirred from about 1 hour to about 24 hours, in embodiments fromabout 2 hours to about 5 hours, to aid in the extraction of the polarsolvent from the microspheres. The microspheres may then be collectedvia filtration and washed (e.g., with n-heptane) to remove any trace ofcontinuous and discontinuous phase liquids on the surface of themicrospheres. The microspheres may then be collected and transferredinto a glass scintillation vial under a nitrogen or argon overlay. Inembodiments, the microspheres may be cross-linked with a cationicsolution and then dried.

In further embodiments, as shown in FIG. 3, doubly-encapsulatedmicrospheres 32, such as those encapsulating microspheres 34, may thenbe further encapsulated in either additional microspheres 30 formed frombiodegradable polymer or the oxidized cellulose, depending on thematerial utilized in the second layer encapsulation. In other words,oxidized cellulose is utilized for every other (e.g., alternate) roundof encapsulation (e.g., microspheres 30 and 34) with adjacent rounds(e.g., microsphere 32) being formed using biodegradable polymers otherthan oxidized cellulose. Thus, in embodiments where dissolved oxidizedcellulose was used in the initial round of encapsulation (e.g., to formthe microsphere 34), biodegradable polymers may be used for the second,(e.g., to form the microsphere 32) fourth, sixth, etc. rounds, and withoxidized cellulose being used in third (e.g., to form the microsphere30), fifth, seventh, etc. rounds. Conversely, in embodiments wherebiodegradable polymers are used in the initial round of encapsulation(e.g., to form the microsphere 34), dissolved oxidized cellulose may beused for the second (e.g., to form the microsphere 32), fourth, sixth,etc. rounds, and with the biodegradable polymers being used in third(e.g., to form the microsphere 30), fifth, seventh, etc. rounds.Subsequent encapsulation using dissolved oxidized cellulose and/orbiodegradable polymers may be carried out in the manner described abovewith respect corresponding encapsulation steps.

Multiple encapsulating microspheres offer several therapeutic advantagessuch as, for example, sequential release of multiple bioactive agents asillustrated in plots 40 and 50 of FIGS. 4 and 5. The plot 40 illustratea release profile of a multi-encapsulated microsphere, e.g., microsphere30, having three unique bioactive agents A, B, and C encapsulated withineach of the microspheres 30, 32, 34, respectively. As the microsphere 30degrades, the bioactive agent A is released, with the release profiledecaying over time corresponding to the degradation of the microsphere30. Thereafter, first encapsulated microsphere 32 begins to degrade,thereby releasing the bioactive agent B. Finally, the third bioactiveagent C is released once the microsphere 34 commences degradation.Release profiles of each of the bioactive agents A, B, and C may betailored by adjusting the amount of the encapsulation material (e.g.,oxidized cellulose and/or biodegradable polymers). In embodiments, therelease profiles may overlap such that one bioactive agent (e.g., A) isreleased concurrently with another bioactive agent (e.g., B). In furtherembodiments, the release profiles of each of the bioactive agents may bediscrete (e.g., not overlapping) based on desired use and therapyrequirements.

The plot 50 illustrates a release profile of a multi-encapsulatedmicrosphere, e.g., microsphere 30, having the same bioactive agent Aencapsulated within each of the microspheres 30, 32, 34. Unlike multiplerelease profiles of distinct bioactive agents A, B, C, encapsulating asingle bioactive agent A provides a burst-like release profile, namely,increased dosages of the bioactive agent A are supplied as each of themicrospheres 30, 32, 34 degrades. In addition, multiple layers providean effective method to further slow-down in the release rate of thebioactive agent.

Multi-encapsulated microspheres provide unique advantages that are notattainable using conventional microspheres that encapsulate one or morebioactive agents in a single biodegradable microsphere. Encapsulatingmultiple bioactive agents in a single microsphere simply provides forsimultaneous release of multiple bioactive agents, rather than for astaggered release profile as illustrated in FIG. 4. With respect to asingle bioactive agent, a single microsphere is further incapable ofproviding burst and/or pulsatile release of bioactive agents during itsdegradation as illustrated in FIG. 5.

Multi-encapsualted microspheres provide for more effective bioactiveagent loading. In embodiments, when a water-soluble hydrophilicbioactive agent is encapsulated in oxidized cellulose as the first layerof encapsulation using an oil-in-oil (o/o) emulsion solvent-evaporationmethod, the water-soluble hydrophilic bioactive agent is not lost in theoil-rich, hydrophobic surroundings. During the second round ofmicroencapsulation, e.g., with an oil in water o/w method, thewater-soluble hydrophilic bioactive agent already has a protectivelayer, which again results in lower bioactive agent loss to the aqueousmedia, resulting in higher bioactive agent loading, following doubleencapsulation. The advantage of more effective bioactive agent loadingis useful for encapsulating highly hydrophilic bioactive agentmolecules.

Multi-encapsualted microspheres further provide for additionalprotection of fragile, i.e. more vulnerable to environmental conditions,bioactive agents (e.g. biologics or protein therapeutics).Multi-encapsulation offers a significant advantage in controlling theirrelease while keeping them active and protected from denaturation. Thisis possible for example when a first layer of encapsulation is put inplace with oxidized cellulose, thus providing a protective barrieragainst any harsh conditions in the second (or subsequent) rounds ofmicroencapsulation. This advantage opens up the possibility of effectiveencapsulation and controlled release of some very fragile biologicaltherapeutics (e.g. protein therapeutics).

With respect to FIG. 2, multi-encapsulation also offers the ability forsimultaneous release of multiple bioactive agents. Bioactive agents A,B, and C may be encapsulated individually in the microspheres 22, whichare then encapsulated in the microsphere 20. This allows the bioactiveagents A, B, and C to release simultaneously, while at the same timeensuring that these molecules do not interact with each other prior torelease. Further, an outer encapsulation may be free of any bioactiveagents and may act as a buffer, preventing release of bioactive agentsuntil the outer encapsulation has biodegraded.

The following Examples are being submitted to illustrate embodiments ofthe present disclosure. These Examples are intended to be illustrativeonly and are not intended to limit the scope of the present disclosure.Also, parts and percentages are by weight unless otherwise indicated. Asused herein, “room temperature” or “ambient temperature” refers to atemperature from about 20° C. to about 25° C.

EXAMPLES Comparative Example 1

Incomplete Dissolution of oxidized cellulose having a degree ofoxidation of 0.6 in a solution including 8% by weight lithium chloride(LiCl) and N-methyl-2-N,N-Dimethylacetamide (DMAc).

About 1.6 grams (g) of LiCl was first dissolved in about 20 milliliters(mL) DMAc to form an 8% LiCl in DMAc solution. About 20 milliliters (mL)of the 8% LiCl in DMAc solution was added to a reactor vessel, and washeated to about 160° C. under argon. About 149 milligrams (mg) ofoxidized cellulose having a degree of oxidation of 0.6 was added to thereactor vessel. The mixture was heated for about 1.17 hours, cooled toambient temperature, and discharged from the reactor vessel. The sampledid not fully dissolve, and was observed to discolor significantly,indicating that further oxidation of the oxidized cellulose hadoccurred.

Comparative Example 2

Incomplete Dissolution of oxidized cellulose having a degree ofoxidation of 0.6 in 8% by weight of LiCl in DMAc solution.

About 20 mL of the 8% LiCl in DMAc solution produced above inComparative Example 1 and about 90 mg of oxidized cellulose having adegree of oxidation of 0.6 were added to a reactor vessel. The mixturewas heated to about 150° C. under argon for about 5.3 hours, cooled toambient temperature, and discharged from the reactor vessel. The sampledid not fully dissolve, and was observed to discolor significantly,indicating further oxidation of the oxidized cellulose occurred.

Comparative Example 3

Pretreatment of oxidized cellulose having a degree of oxidation of 0.6in water.

About 22 mg of oxidized cellulose having a degree of oxidation of 0.6was placed in a reactor vessel and about 0.66 grams of deionized waterwas added thereto. The mixture was stirred for a period of time fromabout 2 minutes to about 3 minutes. The water was then removed in avacuum, and about 20 mL of the 8% LiCl in DMAc solution from ComparativeExample 1 was added to a reactor vessel. The mixture was heated to about155° C. for about 4.6 hours. It was then cooled to ambient temperature,and discharged from the reactor vessel. The sample did not fullydissolve. Thus, pretreatment of the oxidized cellulose in water had nodiscernable effect on dissolution.

Comparative Example 4

Dissolution of cellulose in a solution including 1% by weight of LiCl inN-methyl-2-Pyrrolidinone (NMP) under inert atmosphere.

About 20 mL of the NMP and approximately 80 mg of non-modified cellulosewere added to a reactor vessel. The mixture was heated to about 150° C.under argon for about 6 hours and then cooled to about 110° C. afterwhich approximately 0.2 g of LiCl was added to the reactor vessel. Thereactor vessel was maintained at about 110° C. for an additional hourbefore being cooled to about 80° C. The reactor vessel was maintained atabout 80° C. for about 14.5 hours after which it was observed that thesample had not dissolved and that pieces of non-modified cellulose wereobserved in the reactor vessel indicating that 1% LiCl NMP solution didnot completely dissolve cellulose.

Example 1

Dissolution of oxidized cellulose having a degree of oxidation of 0.6 ina solution including 1% by weight of LiCl in N-methyl-2-pyrrolidinone(NMP).

A 100 mL three-neck round-bottom flask was used as a reactor vessel andwas fitted with a gas inlet, a mechanical stirrer, and a gas outlet,which was then connected to a flow rate monitor. The flask was purgedwith argon for about 5 minutes at a rate of approximately 0.4 liter perminute (L/min), which was measured as approximately 5 bubbles per secondby the flow rate monitor.

About 20 mL of anhydrous NMP was pipetted into the flask, which was thenagain purged with argon. Argon flow was adjusted to a rate ofapproximately 0.2 L/min or from about 2 bubbles per second to about 3bubbles per second, as observed on the flow rate monitor.

A helium line was attached to the flask and the argon flow was stopped.The helium line was inserted into the reactor and submerged below theliquid level, and the helium flow was set at approximately 0.2 L/min tosparge the NMP. After about 45 minutes of sparging, the helium line wasremoved and the argon flow was reinitiated at a rate of about 0.2 L/min.

About 80 mg of oxidized cellulose having a degree of oxidation of 0.6was cut into approximately 0.5 cm×0.5 cm square pieces. Argon flow wastemporarily increased to about 0.4 L/min and the oxidized cellulose wasadded to the flask, after which the argon flow was restored to about 0.2L/min.

The mixture was stirred at about 200 revolutions per minute (rpm). Theflask was heated from about 130° C. to about 135° C. using atemperature-controlled heating mantle. The temperature was maintainedfor about 2 hours under argon as the mixture was stirred. Thereafter,the mixture was cooled to a temperature from about 100° C. to about 110°C.

A scintillation vial was purged with argon in preparation for additionof LiCl. About 0.2 grams of anhydrous LiCl was weighed in the vial.Stirring was temporarily suspended and argon flow was increased to about0.4 L/min while the LiCl was added to the reactor vessel. After additionof the LiCl, the argon flow was restored to about 0.2 L/min. Stirringwas resumed at about 450 rpm for about 5 minutes and then reduced toabout 200 rpm.

Temperature was maintained from about 100° C. to about 110° C. Themixture was visually inspected approximately 5 minutes after addition ofthe LiCl and about every 15 minutes thereafter to determine whetheroxidized cellulose was dissolved. The oxidized cellulose was observed tohave undergone complete dissolution. Heating was terminated and thesolution was cooled to ambient temperature and stirred at about 200 rpm.The solution was then transferred into a scintillation vial under argonand sealed. The solution was stored at ambient conditions.

Example 2

Dissolution of oxidized cellulose having a degree of oxidation of 0.6 ina solution including 1% by weight of LiCl in NMP under ambientatmosphere.

The same process was followed as set forth in Example 1 above, exceptthe dissolution was carried out under ambient atmosphere. Oxidizedcellulose was observed to have undergone complete dissolution.

Example 3

Dissolution of oxidized cellulose having a degree of oxidation of 0.6 ina solution including 1% by weight of LiCl in NMP under ambientatmosphere without helium sparging

The same process was followed as set forth in Example 1 above, exceptthe dissolution was carried out under ambient atmosphere and withouthelium sparging. Oxidized cellulose was observed to have undergonecomplete dissolution.

Molecular weight was determined for the dissolved oxidized cellulose ofExamples 1-3 as summarized in Table 1 below.

TABLE 1 Example Mn (g/mol) 1 2.7 × 10{circumflex over ( )}5 2 1.4 ×10{circumflex over ( )}5 3 1.8 × 10{circumflex over ( )}5

As illustrated in Table 1, dissolved oxidized cellulose of Example 1 hadthe highest molecular weight, whereas the dissolved oxidized celluloseof Examples 2 and 3 had a much lower molecular weight. Without beingbound by any particular theory, it is believed that conductingdissolution under ambient atmosphere degrades the oxidized cellulose,resulting in lower molecular weight.

Example 4

Dissolution of non-modified cellulose in 8% by weight on LiCl in NMPsolution.

The same process was followed as set forth in Example 1 above, exceptabout 80 mg of non-modified cellulose was dissolved, the mixture of thenon-modified cellulose and the solvent was heated from about 145° C. toabout 155° C., and about 1.6 grams of anhydrous LiCl was added to themixture to achieve 8% by weight LiCl in NMP solution since 1% LiClsolution was ineffective as illustrated in Comparative Example 4.Further, after addition of LiCl, the temperature was maintained fromabout 100° C. to about 110° C. for at least one hour. The non-modifiedcellulose was observed to have undergone complete dissolution.

Preparation of a Mobile Phase of 1% by Weight of LiCl in NMP Solutionfor Gel Permeation Chromatography (GPC)

About 1.5 liters (L) of NMP was added to a 2 L volumetric flask, whichwas then loosely capped with a glass stopper. NMP was stirred. About 20grams of LiCl was added to the NMP and was stirred for about 60 minutesuntil it was dissolved. About 0.5 L of NMP was added to the 2 liter markand stirring was stopped. Additional NMP was added to the mark and thesolution was mixed by hand-inverting. A 1 micron polytetrafluoroethylene(PTFE) filter membrane was placed in a filtration apparatus and a vacuumwas applied, which enabled the LiCl in NMP solution to flow through themembrane, thereby filtering the solution. The mobile phase solution wasstored at ambient conditions.

Analysis of Dissolved Oxidized Cellulose of Example 1, Non-ModifiedCellulose of Example 4, and a Standard Sample

Samples of the dissolved oxidized cellulose of Example 1, thenon-modified cellulose of Example 4, and a pullalan standard sample wereseparately filtered through a 1 micron PTFE filter membrane into 3separate high-performance liquid chromatography (HPLC) vials. Inaddition, a combined sample was also prepared by combining about 500microliters (μL) of the dissolved oxidized cellulose of Example 1 andabout 500 μL of the pullalan standard sample (at a concentration ofabout 2 mg/mL) in a single HPLC vial.

All of the samples were subjected to GPC analysis performed using a gelpermeation chromatography system with two 300 millimeter (mm)×7.5 mmcolumns of Polymer Laboratories' PLGEL™ in a series configuration. ADAWN® HELEOS™ II multi-angle laser light scattering system from (WyattTechnology of Santa Barbara, Calif.) was used for absolute molecularweight determination. A refractive index model number OPTILAB® rEX inconjunction with the light scattering detector supplied by WyattTechnology was also used during molecular weight analysis.

GPC was performed at a flow rate of about 1 mL per minute, at atemperature of about 50° C., with an injection volume of about 100 μL.GPC chromatograms of the oxidized cellulose of Example 1 and thenon-modified cellulose of Example 4 are shown in FIGS. 6 and 7,respectively.

Example 5

Dissolution of oxidized cellulose having a degree of oxidation of 0.39in 8% by weight of LiCl in DMAc solution.

About 20 mL of DMAc was added to a reactor vessel under argon, followedby sparging thereof for approximately 10 minutes with helium. About 19mg of oxidized cellulose having a degree of oxidation of 0.39 was addedto the reactor vessel, which was initially heated to about 144° C. Afteraddition of the oxidized cellulose, the temperature was increased toabout 152° C. for approximately 3.2 hours. The reactor vessel was thencooled to about 95° C. and about 1.6 grams of LiCl was added to themixture to form an 8% LiCl in DMAc solution. The mixture was then heatedto about 95° C. for about 45 minutes, then cooled to ambienttemperature. The solution was stirred at ambient temperature forapproximately 64 hours, and discharged from the reactor vessel. Theoxidized cellulose was observed to have undergone complete dissolution.

Example 6

Dissolution of oxidized cellulose having a degree of oxidation of 0.39in a solution including 8.8% by weight of LiCl in NMP.

About 20 mL of NMP was added to the reactor vessel under argon followedby sparging thereof for approximately 1 hour with helium. About 10.2 mgof oxidized cellulose having a degree of oxidation of about 0.39 wasadded to the reactor vessel, which was initially heated to a temperaturefrom about 148° C. to about 154° C. for approximately 2.5 hours. Thereactor vessel was then cooled to about 103° C. and about 1.77 grams ofLiCl was added to the mixture to form an 8.8% LiCl in NMP solution. Themixture was then heated to a temperature from about 103° C. to about105° C. for about 1 hour, then cooled to ambient temperature. Thesolution was stirred at ambient temperature for approximately 24 hours,and discharged from the reactor vessel. The oxidized cellulose wasobserved to have undergone complete dissolution.

Example 7

Dissolution of oxidized cellulose having a degree of oxidation of 0.39in a solution including 1% by weight of LiCl in NMP.

About 20 mL of NMP was added to the reactor vessel under argon followedby sparging thereof for approximately 1 hour with helium. About 11 mg ofoxidized cellulose having a degree of oxidation of about 0.39 was addedto the reactor vessel, which was initially heated to a temperature fromabout 143° C. to about 148° C. for approximately 2 hours. The reactorvessel was then cooled to about 100° C. and about 0.20 grams of LiCl wasadded to the mixture to form a 1% LiCl in NMP solution. The mixture wasthen heated to about 93° C. for about 8 minutes, then cooled to ambienttemperature. The solution was stirred at ambient temperature forapproximately 24 hours, and discharged from the reactor vessel. Theoxidized cellulose was observed to have undergone complete dissolution.

Example 8

Formation of oxidized cellulose microspheres from an oxidized cellulosesolution including 1% by weight of LiCl in N-methyl-2-pyrrolidinone(NMP).

A 600 ml glass beaker was set on a ring stand. A constant-torque mixerwas fitted with a medium-shear impeller, which was inserted into thebeaker. Approximately 200 ml of heavy white mineral oil was added to thebeaker with the mixer set to rotate at approximately 1,500 rpm. About1.7 grams of oxidized cellulose solution (oxidized cellulose in NMP) wasadded drop-wise to the vortex of the stirring mineral oil for about 15minutes until all of the solution was added to the oil to form anemulsion including a plurality of oxidized cellulose microspheres.

About 150 ml of isopropyl myristate was added to the emulsion and themixer speed reduced to approximately 900 rpm and maintained for aboutabout 45 minutes. Thereafter, another 150 ml of isopropyl myristate wasadded to the emulsion such that isopropyl myristate was present at aratio to the oil of about 3:2 and rotations were reduced toapproximately 600 rpm.

The emulsion was stirred from about 2 hours to about 3 hours to extractthe NMP from the oxidized cellulose microspheres. After NMP wasextracted, microspheres were collected by filtration. The microsphereswere then washed with a sufficient volume of n-heptane to remove anytrace of processing oils on the surface of the microspheres. Themicrospheres were dried for about 24 hours. Collected microspheres wereimaged using a Zeiss Leo 435, scanning electron microscope (SEM), whichare shown in FIGS. 8A-B at about 100×, and 250×, respectively. The SEMimages show microspheres having a spherical shape and a smooth outersurface.

Example 9

Formation of 18% by weight (theoretical loading) vitamin B-12 loadedoxidized cellulose microparticles, from a 15% by weight/volume oxidizedcellulose solution including 1% by weight of LiCl inN-methyl-2-pyrrolidinone (NMP).

A discontinuous phase was prepared from the oxidized cellulose solutionof Example 1. About 3 grams of the oxidized cellulose solution wascombined with approximately 100 milligrams of cyanocobalmin (vitaminB-12).

A 1 liter glass beaker was set on a ring stand. A constant-torque mixerwas fitted with a medium-shear impeller, which was inserted into thebeaker. Approximately 300 ml of heavy white mineral oil was added to thebeaker with the mixer set to rotate at approximately 550 rpm. Thesolution of cyanocobalmin and oxidized cellulose was then addeddrop-wise to the vortex of the stirring mineral oil for about 15 minutesuntil all of the solution was added to the oil to form an emulsion.

About 300 ml of cottonseed oil was added to the emulsion. The emulsionwas stirred at approximately 900 rpm for about 60 minutes. Thereafter,another 300 ml of cottonseed oil was added to the emulsion. The emulsionwas again stirred at approximately 900 rpm for about 60 minutes. About100 ml of n-heptane was added to the emulsion.

The emulsion was stirred for about 60 minutes to extract the NMP fromthe oxidized cellulose microparticles. After NMP was extracted,microparticles were collected by filtration. The microparticles werethen washed with a sufficient volume of n-heptane to remove any trace ofprocessing oils on the surface of the microparticles. The microparticleswere dried for about 24 hours.

Collected microparticles were imaged using a Zeiss Leo 435 SEM, whichare shown in FIGS. 9A-B at about 500×, and 1100×, respectively. The SEMimages show microparticles having a textured surface with somemicroparticles having an elongated, rod-like shape and others having asphere-like shape.

Example 10

Formation of 40% by weight bupivacaine free base loaded oxidizedcellulose microparticles, from a 15% by weight/volume oxidized cellulosesolution including 1% by weight of LiCl in N-methyl-2-pyrrolidinone(NMP).

The same process was followed as set forth in Example 9 above, exceptabout 253.5 milligrams of bupivacaine free base was added to theoxidized cellulose solution.

Collected microparticles were imaged using a Zeiss Leo 435 SEM, whichare shown in FIGS. 10A-B at about 50× and 250×, respectively. The SEMimages show microparticles having a spherical shape and a texturedsurface. Without being bound by any particular theory, it is believedthat the rougher surface is caused by the wrapping of the crystals ofbupivacaine free base within the oxidized cellulose microparticles.

Example 11

Formation of 40% by weight bupivacaine HCl loaded oxidized cellulosemicroparticles, from a 15% by weight/volume oxidized cellulose solutionincluding 1% by weight of LiCl in N-methyl-2-pyrrolidinone (NMP).

The same process was followed as set forth in Example 9 above, exceptabout 250.2 milligrams of bupivacaine HCl was added to the oxidizedcellulose solution.

Collected microparticles were imaged using a Zeiss Leo 435 SEM, whichare shown in FIGS. 11A-B at about 50× and 250×, respectively. The SEMimages show microsparticles having an irregular, crystalline shape and atextured surface. Without being bound by any particular theory, it isbelieved that structure of the microparticles is caused by theneedle-like crystalline nature of the active ingredient.

Example 12

Formation of 30% (theoretical and actual measurement) by weight vitaminB-12 loaded oxidized cellulose microspheres, from a 15% by weight/volumeoxidized cellulose solution including 1% by weight of LiCl inN-methyl-2-pyrrolidinone (NMP).

The same process was followed as set forth in Example 9 above, exceptabout 200 milligrams of cyanocobalmin (vitamin B-12) was added to theoxidized cellulose solution.

Collected microparticles were imaged using a Zeiss Leo 435 SEM, whichare shown in FIGS. 13A-B at about 1,000× and 1,700×, respectively. TheSEM images show microspheres having a substantially spherical shape anda smooth outer surface.

Actual loading of the 30% B-12 loaded microspheres was determined usinga SpectraMax M2, a UV-Vis spectrophotometer. Approximately 1 mg of B-12was dissolved in about 10 ml of water and scanned from about 200 nm toabout 800 nm in order to determine maximum absorbance. Maximumabsorbance was measured at approximately 358 nm. A stock solution wasmade with about 10 mg B-12 in 200 ml of water. From this stock solution,serial dilutions were made and a five (5) point standard calibrationcurve was constructed as shown in FIG. 12. About 2.55 mg of the 30% B-12loaded microspheres was dissolved in 10 ml water, then further dilutedto achieve a ratio of microspheres to water of about 1:2. The dilutedsolution was analyzed and measured at an absorbance concentration ofapproximately 0.679 as shown in Table 2 below. Actual loading of vitaminB-12 was measured to be about 31%.

TABLE 2 Sample Conc, Total amt., Weights, Absorbance mg/ml mg % API mgVitamin B12 0.679 0.04 0.79 31.0 2.55 oxidized cellulose microspheres

Example 13

Formation of 25% by weight (theoretical loading) vitamin B-12 loadedoxidized cellulose microspheres from a 15% by weight/volume oxidizedcellulose solution including 1% by weight of LiCl inN-methyl-2-pyrrolidinone (NMP).

The same process was followed as set forth in Example 9 above, exceptabout 150 milligrams of vitamin B-12 was added to the oxidized cellulosesolution.

Collected microparticles were imaged using an Olympus SZX16, a lightmicroscope, which are shown in FIGS. 14A-B at about 600× and 1,000×,respectively. The images show microspheres having a substantiallyspherical shape.

Example 14

Formation of poly-D,L,-lactide (PDLLA) microspheres encapsulatingcis-diamminedichloroplatinum(II) (CDDP) loaded oxidized cellulosemicrospheres.

A 1 liter glass beaker was set on a ring stand. A constant-torque mixerwas fitted with a medium-shear impeller, which was inserted into thebeaker. Approximately 200 ml of heavy white mineral oil was added to thebeaker with the mixer set to rotate at approximately 1,800 rpm.

About 300 milligrams of CDDP was added to about 3 grams of the oxidizedcellulose solution having a concentration of about 15 mg/ml, whichformed a gel. The gel was vortexed for about 30 seconds until a uniformconsistency was achieved and no particles of CDDP were visible.

The gel of CDDP and oxidized cellulose was then added drop-wise to thevortex of the stirring cottonseed and mineral oils for about 15 minutesat about 1,800 rpm, until all of the solution was added to the oil toform an emulsion.

About 200 ml of cottonseed oil were added to the emulsion and the mixingspeed was reduced to about 700 rpm after approximately 1 minute. Afterabout 30 minutes, approximately 200 ml of cottonseed oil was added alongwith about 50 ml of n-heptane and the emulsion was mixed forapproximately 2.5 hours to extract the NMP from the oxidized cellulosemicrospheres. After the NMP was extracted, microspheres were collectedunder vacuum by filtration through Whatman No. 4 filter paper. Themicrospheres were then washed with a sufficient volume of n-heptane toremove any trace of processing oils on the surface of the microspheres.

Collected microspheres were imaged using an Olympus SZX16, a lightmicroscope, which are shown in FIG. 15 at about 1,000×. The light imagesshow microspheres having a substantially spherical shape and a smoothsurface. The microspheres were of yellow color showing CDDPencapsulation.

A 4 liter glass beaker was set on a ring stand and the mixer was fittedwith a high-shear radial impeller above a medium-shear bottom impeller.About 2,500 ml of 1% polyvinyl alcohol (PVA) in water was added to thebeaker and the mixing speed was set to about 1,800 rpm. A solutionhaving a concentration of about 200 mg/ml of PDLLA was prepared bydissolving about 1 gram of PDLLA in about 5 ml of dichloromethane. TheCDDP/oxidized cellulose microspheres were then added to the PDLLAsolution and vortexed to ensure a uniform distribution of themicrospheres in the PDLLA solution thereby forming a suspension.

The suspension was then added to the PVA solution. Mixing was maintainedat about 1,810 rpm for about 5 minutes after which, the speed wasreduced to about 1,150 rpm for about 60 minutes. About 500 ml ofdistilled water was then added to the emulsion to extractdichloromethane from the multi-encapsulated microspheres, namely, PDLLAmicrospheres encapsulating the CDDP/oxidized cellulose microsphere. Themulti-encapsulated microspheres were harvested after about 2.5 hours ofmixing. The microspheres were washed with distilled water to remove alltraces of the PVA. They were then collected off each sieve byfiltration. The collected microspheres were then air-dried for about 24hours.

Collected microspheres were imaged using an Olympus SZX16, a lightmicroscope, which are shown in FIG. 16 at about 1,000×. Microsphereswere also embedded in epoxy and a cross-sectional slice of thereof wasobtained, which was then imaged using a FEI Quanta 600 FEG SEM, which isshown in FIG. 17 at about 1,475×. The images of FIGS. 16 and 17 showlarger PDLLA microspheres encapsulating a plurality of oxidizedcellulose microspheres, which are shown in gold (FIG. 16), which inturn, encapsulate CDDP, which is shown in red (FIG. 17).

CDDP, a water-soluble compound, was successfully encapsulated inmicrospheres formed from solubilized oxidized cellulose using anoil-in-oil (o/o), solvent extraction method. These microspheres werethen encapsulated in polylactide microspheres, using asolid-in-oil-in-water, solvent extraction method. The“microsphere(s)-in-a-microsphere” particles were free-flowing and easilyhandled, no fragility was observed. Since CDDP encapsulation wasconducted without water, sodium chloride was not required, which is usedwhen aqueous systems are employed in encapsulating CDDP to preventtransforming the cis form of CDDP into trans, which is has diminishingbioactive effect.

Example 15

Analysis of degree of oxidation of Oxidized Cellulose of Example 1.

Degree of oxidation of dissolved oxidized cellulose was analyzed usingconductimetric and pH metric titration and compared with the degree ofoxidation of undissolved oxidized cellulose.

Multiple samples from about 90 mg to about 700 mg of undissolvedoxidized cellulose and from about 560 mg to about 4.4 grams of about 16%by weight/volume of oxidized cellulose solution of Example 1 wereprepared. Each of the samples was dissolved in about 15 ml of a sodiumhydroxide (NaOH) solution having molarity (M) from about 0.05 M to about0.5 M. The resulting solutions were titrated with a hydrogen chloride(HCl) solution from about 0.05 M to about 0.5 M on a TIM 845 titrationapparatus, from Radiometer Analytical SAS, Villeurganne Cedex, Franceand conductimetric and pH-metric curves were obtained. A blank titrationwas done in the same conditions to determine the NaOH concentration.

The conductometric titration curves showed the presence of strongalkali, corresponding to the excess of NaOH and a weak alkalicorresponding to the carboxyl content, as shown in an illustrativeconductometric curve of FIG. 18. The characteristic pH-metric curve isshown in the FIG. 19, in which the equivalence point corresponds to theresidual NaOH in the samples.

The degree of oxidation for each sample was calculated using thefollowing formulas (I) and (II):

$\begin{matrix}{{DO} = \frac{162 \times {n({COOH})}}{w - \left( {14 \times {n({COOH})}} \right.}} & (I) \\{{n({COOH})} = {\left( {{V\; 2} - {V\; 1}} \right) \times {C({HCl})}}} & ({II})\end{matrix}$

In which V2 is the volume of HCl in liters obtained by the blanktitration or from the conductometric curve as indicated in FIG. 21; V1is the amount HCl in liters as shown in FIG. 21, or the equivalencepoint from the pH-metric titration of FIG. 22; C is HCl concentration inmoles per liter (Mol/L) an w is the weight of oven-dried sample ofundissolved oxidized cellulose in grams.

The degree of oxidation of non-dissolved oxidized cellulose and fordissolved oxidized cellulose of Example 1 samples are summarized inTable 3 below:

TABLE 3 Undissolved Dissolved Oxidized Oxidized Cellulose Cellulose 0.60.53 0.56 0.52 0.57 0.52 0.6 0.56 0.59 0.6 0.6 0.62 0.59 0.61 0.57 mean0.59 0.52 std dev 0.020 0.006

It will be appreciated that of the above-disclosed and other featuresand functions, or alternatives thereof, may be desirably combined intomany other different systems or applications. Also that variouspresently unforeseen or unanticipated alternatives, modifications,variations or improvements therein may be subsequently made by thoseskilled in the art which are also intended to be encompassed by thefollowing claims. Unless specifically recited in a claim, steps orcomponents of claims should not be implied or imported from thespecification or any other claims as to any particular order, number,position, size, shape, angle, or material.

What is claimed is:
 1. A microsphere comprising: an oxidized cellulose;and at least one bioactive agent.
 2. The microsphere according to claim1, wherein the bioactive agent is selected from the group consisting ofa hydrophilic bioactive agent, a protein bioactive agent, andcombinations thereof.
 3. The microsphere according to claim 1, whereinthe microsphere is formed by: contacting an oxidized cellulose solutioncomprising a solvent with the at least one bioactive agent to form adiscontinuous phase liquid; contacting the discontinuous phase liquidwith a continuous phase liquid to form an emulsion; and contacting theemulsion with a third phase liquid to extract the solvent from theemulsion thereby forming a plurality of microspheres.
 4. The microsphereaccording to claim 3, wherein the third phase liquid is miscible withthe continuous phase liquid and the discontinuous phase liquid.
 5. Themicrosphere according to claim 3, wherein the third phase liquid isselected from the group consisting of isopropyl myristate, hexane,triglycerides and combinations thereof.
 6. The microsphere according toclaim 3, wherein the third phase liquid is present in an amount fromabout 130% by volume to about 170% by volume of the continuous phaseliquid.
 7. The microsphere according to claim 3, further comprisingforming the oxidized cellulose solution by: contacting an oxidizedcellulose with the solvent under an inert atmosphere to form a swelledoxidized cellulose; adjusting the swelled oxidized cellulose mixture toa first temperature; contacting the swelled oxidized cellulose afteradjusting the first temperature with a salt under the inert atmosphereto form an oxidized cellulose solution; and adjusting the oxidizedcellulose solution to a second temperature.
 8. The microsphere accordingto claim 7, wherein the first temperature is from about 115° C. to about145° C. and the second temperature is from about 90° C. to about 120° C.9. The microsphere according to claim 7, wherein the solvent is selectedfrom the group consisting of N,N-Dimethylacetamide,N-methyl-2-pyrrolidinone, and combinations thereof.
 10. The microsphereaccording to claim 7, wherein the salt is selected from the groupconsisting of lithium halides, sodium halides, potassium halides, andcombinations thereof.
 11. The microsphere according to claim 3, whereinthe continuous phase liquid is selected from the group consisting ofplant-based oils, petroleum-based oils, silicone-based oils, andcombinations thereof.
 12. The microsphere according to claim 3, whereinthe third phase liquid is miscible with the continuous phase liquid andimmiscible with the discontinuous phase liquid.
 13. The microsphereaccording to claim 3, wherein the third phase liquid is present in anamount from about 8.33% by volume to about 11.11% by volume of thecontinuous phase liquid.
 14. The microsphere according to claim 1,wherein the at least one bioactive agent is hydrophobic.